Method for treating pathological angiogenesis by administering an antibody that inhibits APLNR

ABSTRACT

The present invention provides apelin receptor (APLNR) modulators that bind to APLNR and methods of using the same. The invention includes APLNR modulators such as antibodies, or antigen-binding fragments thereof, that inhibit or attenuate APLNR-mediated signaling. The invention includes APLNR modulators such as antibodies, or antibody fusion proteins thereof, that activate APLNR-mediated signaling. According to certain embodiments of the invention, the antibodies or antigen-binding fragments or antibody fusion proteins are fully human antibodies that bind to human APLNR with high affinity. The APLNR modulators of the invention are useful for the treatment of diseases and disorders associated with APLNR signaling and/or APLNR cellular expression, such as cardiovascular diseases, angiogenesis diseases, metabolic diseases and fibrotic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/038,202,filed May 20, 2016, which is a US National Stage Application under 35USC § 371 of International Application No. PCT/US2014/066687, filed Nov.20, 2014, which claims the benefit under 35 USC § 119(e) of U.S.Provisional Application No. 61/906,568, filed Nov. 20, 2013, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 7410US04-Sequence.txt,created on Oct. 30, 2018 and containing 126,075 bytes.

FIELD OF THE INVENTION

The present invention relates to apelin receptor (APLNR) modulators thatare antibodies, antibody-fusion proteins or antigen-binding fragmentsthereof, which are specific for human APLNR, and methods of use thereof.

BACKGROUND

Preproapelin is a 77 amino acid protein expressed in the human CNS andperipheral tissues, e.g. lung, heart, and mammary gland. Peptidescomprising C-terminal fragments of varying size of apelin peptide wereshown to activate the G protein—coupled receptor, APJ receptor (nowknown as APLNR) (Habata, et al., 1999, Biochem Biophys Acta 1452:25-35;Hosoya, et al., 2000, JBC, 275(28):21061-67; Lee, et al., 2000, JNeurochem 74:34-41; Medhurst, et al., 2003, J Neurochem 84:1162-1172).Many studies indicate that apelin peptides and analogues conveycardiovascular and angiogenic actions through their interaction with theAPJ receptor (APLNR), such as endothelium-dependent vasodilation(Tatemoto et al., 2001, Regul Pept 99:87-92.

The apelin system appears to play a role in pathophysiologicalangiogenesis. Studies have indicated that apelin may be involved inhypoxia-induced retinal angiogenesis (Kasai et al., 2010, ArteriosclerThromb Vasc Bioi 30:2182-2187). In some reports, certain compositionsmay inhibit angiogenesis by inhibiting the apelin/APJ pathway (see,e.g., U.S. Pat. No. 7,736,646), such as APLNR inhibitors capable ofblocking pathological angiogenesis and therefore useful in inhibitingtumor growth or vascularization in the retina (Kojima, Y. andQuertermous, T., 2008, Arterioscler Thromb Vasc Biol; 28; 1687-1688;Rayalam, S. et al. 2011, Recent Pat Anticancer Drug Discov 6(3):367-72).As such, interference with apelin-mediated signaling may also bebeneficial in early prevention of proliferative diabetic retinopathy(Tao et al., 2010, Invest Opthamol Visual Science 51:4237-4242; Lu, Q.et al, 2013, PLoS One 8(7):e69703).

Apelin has also been reported in the regulation of insulin andmechanisms of diabetes and obesity-related disorders. In mouse models ofobesity, apelin is released from adipocytes and is directly upregulatedby insulin (Boucher, et al., 2005, Endocrinol 146:1764-71). Apelinknockout mice demonstrate diminished insulin sensitivity (Yue, et al.,2010, Am J Physiol Endocrinol Metab 298:E59-E67).

Furthermore, apelin-induced vasodilation and angiogenesis may beprotective in ischemia-reperfusion injury and improve cardiac functionin conditions such as congestive heart failure, myocardial infarction,and cardiomyopathy. Therapeutic administration of apelin peptidesreportedly contributes to the promotion of angiogenesis and functionalrecovery from ischemia. (Eyries M, et al., 2008, Circ Res 103:432-440;Kidoya H, et al., 2010, Blood 115:3166-3174).

APLNR signaling, and modulation thereof, has been implicated as a factorin a variety of diseases and disorders (e.g. WO2004081198A2, publishedon 23 Sep. 2004), and there is still a need for therapeutic agents thatmodulate APLNR biological activity.

BRIEF SUMMARY OF THE INVENTION

The present invention provides APLNR modulators that bind human apelinreceptor (“APLNR”). The APLNR modulators of the invention are useful,inter alia, for activating or inhibiting APLNR-mediated signaling andfor treating diseases and disorders related to APLNR activity and/orsignaling.

The APLNR modulators of the invention include antibodies,antibody-fusion proteins, and antigen-binding fragments thereof.

The antibodies and antibody-fusion proteins of the invention can befull-length (for example, an IgG1, IgG2 or IgG4 antibody) or maycomprise only an antigen-binding portion (for example, a Fab, F(ab′)₂ orscFv fragment), and may be modified to affect functionality, e.g., toeliminate residual effector functions (Reddy et al., 2000, J. Immunol.164:1925-1933).

The present invention provides antibodies, antibody-fusion proteins orantigen-binding fragments thereof comprising a heavy chain variableregion (HCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, and 210, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment of an antibody comprising a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138,154, 170, 186, 202, and 218, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment thereof comprising a HCVR and LCVR(HCVR/LCVR) sequence pair selected from the group consisting of SEQ IDNO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154, 162/170, 178/186, 194/202, and 210/218.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment of an antibody comprising a heavy chain CDR3(HCDR3) domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168,184, 200, and 216, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a light chain CDR3 (LCDR3) domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96,112, 128, 144, 160, 176, 192, 208, and 224, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

In certain embodiments, the antibody, antibody-fusion protein orantigen-binding portion of an antibody comprises a HCDR3/LCDR3 aminoacid sequence pair selected from the group consisting of SEQ ID NO:8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 120/128, 136/144,152/160, 168/176, 184/192, 200/208, and 216/224.

The present invention also provides an antibody, antibody-fusion proteinor fragment thereof further comprising a heavy chain CDR1 (HCDR1) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, and212, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a heavy chainCDR2 (HCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134,150, 166, 182, 198, and 214, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a light chain CDR1 (LCDR1) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 12, 28, 44,60, 76, 92, 108, 124, 140, 156, 172, 188, 204, and 220, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a light chainCDR2 (LCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142,158, 174, 190, 206, and 222, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Certain non-limiting, exemplary antibodies, antibody-fusion proteins andantigen-binding fragments of the invention compriseHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having theamino acid sequences selected from the group consisting of: SEQ ID NOs:4-6-8-12-14-16 (e.g. H1M9207N); 20-22-24-28-30-32 (e.g. H2aM9209N);36-38-40-44-46-48 (e.g. H2aM9222N); 52-54-56-60-62-64 (e.g. H2aM9227N);68-70-72-76-78-80 (e.g. H2aM9228N); 84-86-88-92-94-96 (e.g. H2aM9230N);100-102-104-108-110-112 (e.g. H2aM9232N); 116-118-120-124-126-128 (e.g.H4H9092P); 132-134-136-140-142-144 (e.g. H4H9093P);148-150-152-156-158-160 (e.g., H4H9101P); 164-166-168-172-174-176 (e.g.H4H9103P); 180-182-184-188-190-192 (e.g., H4H9104P);196-198-200-204-206-208 (e.g. H4H9112P); and 212-214-216-220-222-224(e.g. H4H9113P).

In a related embodiment, the invention includes an antibody,antibody-fusion protein or antigen-binding fragment of an antibody whichspecifically binds APLNR, wherein the antibody, antibody-fusion proteinor antigen-binding fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain variable region(HCVR/LCVR) sequences selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138,146/154, 162/170, 178/186, 194/202, and 210/218. Methods and techniquesfor identifying CDRs within HCVR and LCVR amino acid sequences are wellknown in the art and can be used to identify CDRs within the specifiedHCVR and/or LCVR amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., 1997, J. Mol. Biol.273:927-948; and Martin et al., 1989, Proc. Natl. Acad. Sci. USA86:9268-9272. Public databases are also available for identifying CDRsequences within an antibody.

The present invention also provides an antibody-fusion protein orfragment thereof further comprising an apelin peptide. Certainnon-limiting, exemplary antibody-fusion proteins of the inventioncomprise heavy and light chain variable region (HCVR/LCVR) sequencesselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, and 210/218; and further comprise an apelin peptidesequence, e.g. a fragment or analogue of SEQ ID NO: 227, SEQ ID NO: 228,SEQ ID NO: 229 or SEQ ID NO: 230. In certain embodiments, the apelinpeptide sequence, or fragment or analogue thereof, comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 227, SEQID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 262, SEQ ID NO:269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQID NO: 283, SEQ ID NO: 284, and SEQ ID NO: 285.

The present invention provides antibody-fusion proteins orantigen-binding fragments thereof comprising a heavy chain (HC) havingan amino acid sequence selected from the group consisting of SEQ ID NO:239, 241, 243, 245, 247, 253, 255, 257, 259, 274, 275, 276, 277, 278,279, 280, 281, and 282, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

The present invention also provides an antibody-fusion protein orantigen-binding fragment of an antibody-fusion protein comprising alight chain (LC) having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 235, 237, 249, and 251, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides an antibody-fusion protein orantigen-binding fragment thereof comprising a HC and LC (HC/LC) aminoacid sequence pair selected from the group consisting of SEQ ID NO:130/235, 130/237, 239/138, 241/138, 243/138, 245/138, 247/122, 114/249,114/251, 253/26, 255/26, 257/26, 259/26, 274/138, 275/138, 276/138,277/138, 278/138, 279/26, 280/26, 281/26, and 282/26.

Certain non-limiting, exemplary antibody-fusion proteins comprise (i) animmunoglobulin (Ig) molecule and (ii) an apelin peptide, or analoguethereof. In some embodiments, the IgG molecule is an anti-APLNR antibodyas described herein. In further embodiments, the apelin peptidecomprises the amino acid sequence selected from the group consisting ofSEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229 or SEQ ID NO: 230, orcomprises a fragment or analogue of the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO:229 or SEQ ID NO: 230.

Another aspect of the invention provides a protein comprisingN′-P1-X1(n)-A1-C′ or N′-A1-X1 (n)-P1-C′, wherein N′ is the N-terminusand C′ is the C-terminus of the polypeptide; P1 comprises an amino acidsequence selected from the group consisting of an HCVR, an LCVR, a heavychain, a light chain, and an HCVR/LCVR ScFv sequence; A1 comprises anapelin peptide, or an analogue thereof; and X1 is a peptide linker;wherein n=0 to 10.

In some embodiments, the apelin peptide, or analogue thereof comprisesapelin40-77 (apelin-38), apelin42-77 (apelin-36), apelin43-77(apelin-35), apelin47-77 (apelin-31), apelin59-77 (apelin-19),apelin61-77 (apelin-17), apelin63-77 (apelin-15), apelin64-77(apelin-14), apelin65-77 (apelin-13), apelin66-77 (apelin-12, or A12),apelin67-77 (apelin-11), apelin68-77 (apelin-10), apelin73-77(apelin-5), apelin61-76 (apelin-K16P), apelin61-75 (apelin-K15M),apelin61-74 (apelin-K14P), or [Pyr¹]Apelin-13.

According to certain embodiments, the antibody-fusion protein orantigen-binding fragment thereof comprises the heavy and light chainsequences encoded by the amino acid sequences of SEQ ID NOs: 130 and 235(e.g. H4H9093P-1-NVK3), 130 and 237 (e.g. H4H9093P-2-CVK3), 239 and 138(e.g. H4H9093P-3-NVH3), 241 and 138 (e.g. H4H9093P-4-NVH0), 243 and 138(e.g. H4H9093P-5-NVH1), 245 and 138 (e.g. H4H9093P-6-NVH2), 247 and 122(e.g. H4H9092P-1-NVH3), 114 and 249 (e.g. H4H9092P-2-NVK3), 114 and 251(e.g. H4H9092P-3-CVK3), 253 and 26 (e.g. H4H9209N-1-NVH0), 255 and 26(e.g. H4H9209N-2-NVH1), 257 and 26 (e.g. H4H9209N-3-NVH2), 259 and 26(e.g. H4H9209N-4-NVH3), 274 and 138 (e.g. H4H9093P-APN9-(G4S)3), 275 and138 (e.g. H4H9093P-APN10-(G4S)3), 276 and 138 (e.g.H4H9093P-APN11-(G4S)3), 277 and 138 (e.g. H4H9093P-APN11+S-(G4S)3), 278and 138 (e.g. H4H9093P-APNV5-11-(G4S)3), 279 and 26 (e.g.H4H9209N-APN9-(G4S)3), 280 and 26 (e.g. H4H9209N-APN10-(G4S)3), 281 and26 (e.g. H4H9209N-APN11-(G4S)3), or 282 and 26 (e.g.H4H9209N-APN11+S-(G4S)3).

In another aspect, the invention provides nucleic acid moleculesencoding anti-APLNR antibodies, antibody-fusion proteins orantigen-binding fragments thereof. Recombinant expression vectorscarrying the nucleic acids of the invention, and host cells into whichsuch vectors have been introduced, are also encompassed by theinvention, as are methods of producing the antibodies or antibody-fusionproteins by culturing the host cells under conditions permittingproduction of the antibodies, and recovering the antibodies orantibody-fusion proteins produced.

In one embodiment, the invention provides an antibody, antibody-fusionprotein or antigen-binding fragment thereof comprising a HCVR encoded bya nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, and 209, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment thereof comprising a LCVR encoded by anucleic acid sequence selected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, and 217, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment of an antibody comprising a HCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, and215, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207,and 223, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody, antibody-fusion proteinor antigen-binding fragment thereof which further comprises a HCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163,179, 195, and 211, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof; aHCDR2 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165,181, 197, and 213, or a substantially identical sequence having at least90%, at least 95%, at least 98%, or at least 99% homology thereof; aLCDR1 domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155,171, 187, 203, and 219, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof;and a LCDR2 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141,157, 173, 189, 205, and 221, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%homology thereof.

According to certain embodiments, the antibody, antibody-fusion proteinor antigen-binding fragment thereof comprises the heavy and light chainCDR sequences encoded by the nucleic acid sequences of SEQ ID NOs: 1 and9 (e.g. H1M9207N), 17 and 25 (e.g. H2aM9209N), 33 and 41 (e.g.H2aM9222N), 49 and 57 (e.g. H2aM9227N), 65 and 73 (e.g. H2aM9228N), 81and 89 (e.g. H2aM9230N), 97 and 105 (e.g. H2aM9232N), 113 and 121 (e.g.H4H9092P), 129 and 137 (e.g. H4H9093P), 145 and 153 (e.g. H4H9101P), 161and 169 (e.g. H4H9103P), 177 and 185 (e.g. H4H9104P), 193 and 201 (e.g.H4H9112P), or 209 and 217 (e.g. H4H9113P).

In one embodiment, the invention provides an antibody-fusion protein orantigen-binding fragment thereof comprising a heavy chain (HC) encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO: 238, 240, 242, 244, 246, 252, 254, 256, and 258, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof.

The present invention also provides an antibody-fusion protein orantigen-binding fragment thereof comprising a light chain (LC) encodedby a nucleic acid sequence selected from the group consisting of SEQ IDNO: 234, 236, 248, and 250, or a substantially identical sequence havingat least 90%, at least 95%, at least 98%, or at least 99% homologythereof.

According to certain embodiments, the antibody-fusion protein orantigen-binding fragment thereof comprises the heavy and light chainsequences encoded by the nucleic acid sequences of SEQ ID NOs: 129 and234 (e.g. H4H9093P-1-NVK3), 129 and 236 (e.g. H4H9093P-2-CVK3), 238 and137 (e.g. H4H9093P-3-NVH3), 240 and 137 (e.g. H4H9093P-4-NVH0), 242 and137 (e.g. H4H9093P-5-NVH1), 244 and 137 (e.g. H4H9093P-6-NVH2), 246 and121 (e.g. H4H9092P-1-NVH3), 113 and 248 (e.g. H4H9092P-2-NVK3), 113 and250 (e.g. H4H9092P-3-CVK3), 252 and 25 (e.g. H4H9209N-1-NVH0), 254 and25 (e.g. H4H9209N-2-NVH1), 256 and 25 (e.g. H4H9209N-3-NVH2), or 258 and25 (e.g. H4H9209N-4-NVH3).

In other embodiments, the antibody-fusion protein comprises nucleic acidmolecules encoding the heavy and light chain amino acid pairs selectedfrom the group consisting of SEQ ID NOs: 130 and 235 (e.g.H4H9093P-1-NVK3), 130 and 237 (e.g. H4H9093P-2-CVK3), 239 and 138 (e.g.H4H9093P-3-NVH3), 241 and 138 (e.g. H4H9093P-4-NVH0), 243 and 138 (e.g.H4H9093P-5-NVH1), 245 and 138 (e.g. H4H9093P-6-NVH2), 247 and 122 (e.g.H4H9092P-1-NVH3), 114 and 249 (e.g. H4H9092P-2-NVK3), 114 and 251 (e.g.H4H9092P-3-CVK3), 253 and 26 (e.g. H4H9209N-1-NVH0), 255 and 26 (e.g.H4H9209N-2-NVH1), 257 and 26 (e.g. H4H9209N-3-NVH2), 259 and 26 (e.g.H4H9209N-4-NVH3), 274 and 138 (e.g. H4H9093P-APN9-(G4S)3), 275 and 138(e.g. H4H9093P-APN10-(G4S)3), 276 and 138 (e.g. H4H9093P-APN11-(G4S)3),277 and 138 (e.g. H4H9093P-APN11+S-(G4S)3), 278 and 138 (e.g.H4H9093P-APNV5-11-(G4S)3), 279 and 26 (e.g. H4H9209N-APN9-(G4S)3), 280and 26 (e.g. H4H9209N-APN10-(G4S)3), 281 and 26 (e.g.H4H9209N-APN11-(G4S)3), or 282 and 26 (e.g. H4H9209N-APN11+S-(G4S)3).

According to other embodiments, the invention provides a firstpolynucleotide and a second polynucleotide which together encode anantibody-fusion protein. The invention further provides a cellcomprising a first polynucleotide and a second polynucleotide whichtogether encode an antibody-fusion protein. In some embodiments, thefirst and second polynucleotides are a part of the same nucleic acidmolecule or different nucleic acid molecules in the cell.

In certain examples, the first polynucleotide encodes a polypeptidecomprising (i) a heavy chain variable region (HCVR) having an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, and 210, and (ii) anapelin peptide which is a fragment or analogue of the preproapelinpolypeptide having an amino acid sequence of SEQ ID NO: 227; and thesecond polynucleotide encodes a polypeptide comprising a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138,154, 170, 186, 202, and 218.

In other embodiments, the first polynucleotide encodes a polypeptidecomprising a heavy chain variable region (HCVR) having an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,50, 66, 82, 98, 114, 130, 146, 162, 178, 194, and 210, and the secondpolynucleotide encodes a polypeptide comprising (i) a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138,154, 170, 186, 202, and 218, and (ii) an apelin peptide which is afragment or analogue of the preproapelin polypeptide having an aminoacid sequence of SEQ ID NO: 227.

The present invention includes anti-APLNR antibodies and antibody-fusionproteins having a modified glycosylation pattern. In some applications,modification to remove undesirable glycosylation sites may be useful, oran antibody lacking a fucose moiety present on the oligosaccharidechain, for example, to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al., 2002, JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody, antibody-fusion protein orfragment thereof which specifically binds APLNR and a pharmaceuticallyacceptable carrier. In a related aspect, the invention features acomposition which is a combination of an anti-APLNR antibody orantibody-fusion protein and a second therapeutic agent. In oneembodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-APLNR antibody or antibody-fusionprotein. Exemplary agents that may be advantageously combined with ananti-APLNR antibody include, without limitation, other agents thatinhibit APLNR activity (including other antibodies or antigen-bindingfragments thereof, fusion proteins, peptide agonists or antagonists,small molecules, etc.) and/or agents which do not directly bind APLNRbut nonetheless interfere with, block or attenuate APLNR-mediatedsignaling. Exemplary agents that may be advantageously combined with anantibody-fusion protein include, without limitation, other agents thatactivate APLNR activity (including other fusion proteins, antibodies orantigen-binding fragments thereof, peptide agonists or antagonists,small molecules, etc.) and/or agents which activate APLNR signaling ordownstream cellular effects. Additional combination therapies andco-formulations involving the antibodies and antibody-fusion proteins ofthe present invention are disclosed elsewhere herein. As such, apharmaceutical composition is provided comprising any one or more of theantibodies, antibody-fusion proteins or antigen-binding fragmentsthereof, in accordance with the invention.

In yet another aspect, the invention provides therapeutic methods forinhibiting APLNR activity using an APLNR modulator of the invention,wherein the therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibody,antibody-fusion protein or antigen-binding fragment of an antibody ofthe invention. The disorder treated is any disease or condition which isimproved, ameliorated, inhibited or prevented by removal, inhibition orreduction of APLNR activity or signaling. The anti-APLNR antibodies,antibody-fusion proteins or antibody fragments of the invention mayfunction to block the interaction between APLNR and an APLNR bindingpartner (e.g., an APLNR receptor ligand such as an apelin peptide), orotherwise inhibit the signaling activity of APLNR.

In still another aspect, the invention provides therapeutic methods foractivating APLNR activity using an APLNR modulator of the invention,wherein the therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibody,antibody-fusion protein or antigen-binding fragment thereof of theinvention. The disorder treated is any disease or condition which isimproved, ameliorated, inhibited or prevented by activation,stimulation, or amplification of APLNR activity or signaling. Theanti-APLNR antibodies, antibody-fusion proteins or antibody fragments ofthe invention may function to enhance the interaction between APLNR andan APLNR binding partner (e.g., an APLNR receptor ligand such as anapelin peptide), or otherwise activate or augment the signaling activityof APLNR.

The present invention also includes the use of an anti-APLNR antibody,antibody-fusion protein or antigen-binding portion of an antibody of theinvention in the manufacture of a medicament for the treatment of adisease or disorder related to or caused by APLNR activity in a patient.The invention further provides an antibody composition for use in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by APLNR activity in a patient, such disease ordisorder selected from the group consisting of cardiovascular disease,acute decompensated heart failure, congestive heart failure, myocardialinfarction, cardiomyopathy, ischemia, ischemia/reperfusion injury,pulmonary hypertension, diabetes, neuronal injury, neurodegeneration,hot flash symptoms, fluid homeostasis, HIV infection, obesity, cancer,metastatic disease, retinopathy, fibrosis, and pathologicalangiogenesis.

The invention further provides a method for treating cardiovasculardisease, acute decompensated heart failure, congestive heart failure,myocardial infarction, cardiomyopathy, ischemia, ischemia/reperfusioninjury, pulmonary hypertension, diabetes, neuronal injury,neurodegeneration, hot flash symptoms, fluid homeostasis, HIV infection,obesity, cancer, metastatic disease, retinopathy, fibrosis, orpathological angiogenesis, the method comprising administering apharmaceutical composition comprising any of the antibodies,antibody-fusion proteins or antigen-binding fragments thereof, accordingto the invention, to a subject in need thereof.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the statistical analysis of the effects of anti-APLNRantibody in an RVD model. An antagonistic anti-APLNR antibody,H2aM9232N, produced a statistically significant mean reduction ofapproximately 30% in retinal blood vessel outgrowth compared to control(hFc) in the developing mouse retina, indicating that APLNR blockade hasa significant anti-angiogenic effect (**p<0.005; two-tailed pvalue=0.0014; t=4.123,df=12).

FIG. 2 depicts the pattern of intact apelin peptide peaks on massspectrometry after 0, 6 and 24 hours of exposure to serum for truncatedapelin fusion antibodies, H4H9209N-APN11-(G4S)3 (FIG. 2A) orH4H9209N-APN11+S-(G4S)3 (FIG. 2B). The peptide of interest, after Lys-Cdigestion of the fusion antibody after serum exposure, has the sequenceof QRPRLSHK, reporting a mass charge ratio peak at 1004. Theapelin-cter11 fusion antibody has residual apelin peak after 24 hours ofserum exposure.

FIG. 3 shows activity of the Apelin-antibody fusions (H4H9093P-3-NVH3,H4H9209N-APN11-(G4S)3, or H4H9209N-APN11+S-(G4S)3) exposed to dilutedserum in a beta-arrestin activity assay (DiscoverX β-Arrestin activityassay) at timepoints 0, 6 and 24 hours. Antibody fusions havingApelin-Cter11 and apelin-Cter11+S at their C-termini retain β-Arrestinactivity after 6 h of serum exposure. The 6 h timepoint value representspercent activation relative to the 0 h timepoint, or 2.4%, 70.4% and33.6% for H4H9093P-3-NVH3, H4H9209N-APN11-(G4S)3, orH4H9209N-APN11+S-(G4S)3, respectively.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expressions “apelin receptor,” “APLNR,” “APJ receptor,” and thelike, as used herein, refer to a human APLNR protein having the aminoacid sequence of SEQ ID NO: 225, or a substantially similar amino acidsequence to SEQ ID NO: 225. All references to proteins, polypeptides andprotein fragments herein are intended to refer to the human version ofthe respective protein, polypeptide or protein fragment unlessexplicitly specified as being from a non-human species (e.g., “mouseAPLNR,” “monkey APLNR,” etc.).

As used herein, “an antibody or antibody-fusion protein that bindsAPLNR” or an “anti-APLNR antibody” includes immunoglobulin molecules,antibodies, antibody-fusion proteins and antigen-binding fragmentsthereof that bind a soluble fragment of an APLNR protein. Soluble APLNRmolecules include natural APLNR proteins as well as recombinant APLNRprotein variants such as, e.g., monomeric and dimeric APLNR constructs.

The term “immunoglobulin” (Ig) refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) chains and one pair of heavy (H) chains, which may all four beinter-connected by disulfide bonds. The structure of immunoglobulins hasbeen well characterized. See for instance Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). The proteins of theinvention comprise amino acid sequences that may be derived from animmunoglobulin molecule, such as derived from any immunoglobulin regionor domain.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., APLNR). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM), as well as immunoglobulin moleculesincluding a fragment of one or more heavy chains or a fragment of one ormore light chains, (e.g. Fab, F(ab′)₂ or scFv fragments), as describedherein. Each heavy chain comprises a heavy chain variable region(abbreviated herein as HCVR or V_(H)) and a heavy chain constant region.The heavy chain constant region comprises three domains, C_(H)1, C_(H)2and C_(H)3. Each light chain comprises a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region comprises one domain (C_(L)1). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-APLNR antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc. Suchtechniques may also be employed to synthesize any antibody-fusionmolecule containing an antigen-binding fragment derived from a fullantibody molecule.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)—V_(H), V_(H)—V_(L) orV_(L)—V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; V_(H)—C_(H)2-C_(H)3; V_(H)—C_(L); (viii)V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The phrase “antibody-fusion proteins” includes recombinant polypeptidesand proteins derived from antibodies of the invention that have beenengineered to contain an antibody or antigen-binding fragment asdescribed herein. For example, an “antibody-apelin fusion protein”includes a chimeric protein comprising an amino acid sequence derivedfrom an anti-APLNR antibody fused to an amino acid sequence of an apelinpeptide or analogue. The apelin peptide component may be fused to theanti-APLNR antibody or antigen-binding fragment either at the N-terminusor the C-terminus of the antibody light chain or heavy chain, with orwithout peptide linkers. The phrase “fused to”, as used herein, means(but is not limited to) a polypeptide formed by expression of a chimericgene made by combining more than one sequence, typically by cloning onegene into an expression vector in frame with a second gene such that thetwo genes are encoding one continuous polypeptide. Recombinant cloningtechniques, such as polymerase chain reaction (PCR) and restrictionendonuclease cloning, are well-known in the art. In addition to beingmade by recombinant technology, parts of a polypeptide can be “fused to”each other by means of chemical reaction, or other means known in theart for making custom polypeptides.

In some embodiments, the components or amino acids of an antibody-fusionprotein are separated by a linker (or “spacer”) peptide. Such peptidelinkers are well known in the art (e.g., polyglycine or Gly-Ser linkers)and typically allow for proper folding of one or both of the componentsof the antibody-fusion protein. The linker provides a flexible junctionregion of the component of the fusion protein, allowing the two ends ofthe molecule to move independently, and may play an important role inretaining each of the two moieties' appropriate functions. Therefore,the junction region acts in some cases as both a linker, which combinesthe two parts together, and as a spacer, which allows each of the twoparts to form its own biological structure and not interfere with theother part. Furthermore, the junction region should create an epitopethat will not be recognized by the subject's immune system as foreign,in other words, will not be considered immunogenic. Linker selection mayalso have an effect on binding activity, and thus the bioactivity, ofthe fusion protein. (See Huston, et al, 1988, PNAS, 85:16:5879-83;Robinson & Bates, 1998, PNAS 95(11):5929-34; and Arai, et al. 2001,PEDS, 14(8):529-32; Chen, X. et al., 2013, Advanced Drug DeliveryReviews 65:1357-1369.) In one embodiment, the apelin peptide isconnected to the C-terminus or to the N-terminus of the light chain orheavy chain of the antibody or antigen-binding fragment thereof, via oneor more peptide linkers.

The antibodies and antibody-fusion proteins of the present invention mayfunction through complement-dependent cytotoxicity (CDC) orantibody-dependent cell-mediated cytotoxicity (ADCC).“Complement-dependent cytotoxicity” (CDC) refers to lysis ofantigen-expressing cells by an antibody of the invention in the presenceof complement. “Antibody-dependent cell-mediated cytotoxicity” (ADCC)refers to a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland thereby lead to lysis of the target cell. CDC and ADCC can bemeasured using assays that are well known and available in the art.(See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al.,1998, Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region ofan antibody or antibody-fusion protein is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

In another aspect, the antibody or antibody-fusion protein may beengineered at its Fc domain to activate all, some, or none of the normalFc effector functions, without affecting the antibody's desiredpharmacokinetic properties. Therefore, antibodies or antibody-fusionproteins with engineered Fc domains that have altered Fc receptorbinding may have reduced side effects. Thus, in one embodiment, theprotein comprises a chimeric or otherwise modified Fc domain. For anexample of a chimeric Fc domain, see PCT International Publication No.WO/2014/121087 A1, published Aug. 7, 2014, which is herein incorporatedby reference in its entirety.

In certain embodiments of the invention, the anti-APLNR antibodies andantibody-fusion proteins of the invention are human antibodies. The term“human antibody”, as used herein, is intended to include antibodieshaving variable and constant regions derived from human germlineimmunoglobulin sequences. The human antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The antibodies and antibody-fusion proteins of the invention may, insome embodiments, be recombinant human antibodies. The term “recombinanthuman antibody”, as used herein, is intended to include all humanantibodies and fusion proteins thereof that are prepared, expressed,created or isolated by recombinant means, such as antibodies expressedusing a recombinant expression vector transfected into a host cell(described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al., 1992, Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.,1993, Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies and antibody-fusion proteins of the invention may beisolated antibodies. An “isolated antibody,” as used herein, means anantibody that has been identified and separated and/or recovered from atleast one component of its natural environment. For example, an antibodythat has been separated or removed from at least one component of anorganism, or from a tissue or cell in which the antibody naturallyexists or is naturally produced, is an “isolated antibody” for purposesof the present invention. An isolated antibody also includes an antibodyin situ within a recombinant cell. Isolated antibodies are antibodiesthat have been subjected to at least one purification or isolation step.According to certain embodiments, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The present invention includes neutralizing and/or blocking anti-APLNRantibodies and antibody-fusion proteins. A “neutralizing” or “blocking”antibody, as used herein, is intended to refer to an antibody whosebinding to APLNR: (i) interferes with the interaction between APLNR oran APLNR fragment and an APLNR receptor component (e.g., apelin peptide,etc.); and/or (ii) results in inhibition of at least one biologicalfunction of APLNR. The inhibition caused by an APLNR neutralizing orblocking antibody need not be complete so long as it is detectable usingan appropriate assay.

The term “antagonist”, as used herein, refers to a moiety that binds tothe receptor at the same site or near the same site as an agonist (forexample, the endogenous ligand), but which does not activate theintracellular response typically initiated by the active form of thereceptor, and thereby inhibits or neutralizes the intracellular responseby an agonist or partial agonist. In some cases, antagonists do notdiminish the baseline intracellular response in the absence of anagonist or partial agonist. An antagonist does not necessarily have tofunction as a competitive binding inhibitor, but may work bysequestering an agonist, or indirectly modulating a downstream effect.

The present invention includes anti-APLNR antibodies and antibody-fusionproteins that activate APLNR, however to a lesser extent than theactivation exhibited by a full agonist of the APLNR, such as an apelinpeptide. For example, such an “activating” antibody, as used herein, isintended to refer to an antibody whose binding to APLNR: (i) augmentsthe interaction between APLNR or an APLNR fragment and an APLNR ligand(e.g., apelin peptide, etc.); and/or (ii) results in activation of atleast one biological function of APLNR. The activation caused by ananti-APLNR antibody need not be complete so long as it is detectableusing an appropriate assay. To this end, an activating antibody mayfunction as a partial or inverse agonist of the APLNR.

The term “agonist”, as used herein, refers to a moiety that interactswith (directly or indirectly binds) and activates the receptor andinitiates a physiological or pharmacological response characteristic ofthat receptor, such as when bound to its endogenous ligand. For example,upon binding to APLNR, apelin activates the receptor which internalizesthe receptor. Also, APLNR-apelin binding activates APLNR which decreasesadenylyl cyclase activity and therefore inhibits cAMP accumulation inthe cell.

The term “EC₅₀” or “EC50”, as used herein, refers to the half maximaleffective concentration, which includes the concentration of a ligandthat induces a response, for example a cellular response, halfwaybetween the baseline and maximum after a specified exposure time. TheEC₅₀ essentially represents the concentration of a ligand where 50% ofits maximal effect is observed. Thus, with regard to cellular signaling,increased receptor activity is observed with a decreased EC₅₀ value,i.e. half maximal effective concentration value (less ligand needed toproduce a greater response).

The term “IC₅₀” or “IC50”, as used herein, refers to the half maximalinhibitory concentration of a cellular response. In other words, themeasure of the effectiveness of a particular moiety (e.g. protein,compound, or molecule) in inhibiting biological or biochemical receptorfunction, wherein an assay quantitates the amount of such moiety neededto inhibit a given biological process. Thus, with regard to cellularsignaling, a greater inhibitory activity is observed with a decreasedIC₅₀ value.

Exemplary assays for detecting APLNR activation and inhibition aredescribed in the working Examples herein.

The anti-APLNR antibodies and antibody-fusion proteins disclosed hereinmay comprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes antibodiesand antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies and antibody-fusion proteins of the present invention maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germlinesequence while certain other residues that differ from the originalgermline sequence are maintained or are mutated to the correspondingresidue of a different germline sequence. Once obtained, antibodies, andantibody-fusion proteins and antigen-binding fragments that contain oneor more germline mutations can be easily tested for one or more desiredproperty such as, improved binding specificity, increased bindingaffinity, improved or enhanced antagonistic or agonistic biologicalproperties (as the case may be), reduced immunogenicity, etc.Antibodies, and antibody-fusion proteins and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-APLNR antibodies andantibody-fusion proteins comprising variants of any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein having one or moreconservative substitutions. For example, the present invention includesanti-APLNR antibodies having HCVR, LCVR, and/or CDR amino acid sequenceswith, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc.conservative amino acid substitutions relative to any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson, 1994,Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al., 1992, Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, 1994, supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al.,1997, Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Biological Characteristics of the APLNR Modulators

The present invention includes anti-APLNR antibodies and antigen-bindingfragments thereof that bind human APLNR and inhibit or attenuateAPLNR-mediated signaling. An anti-APLNR antibody is deemed to “inhibitor attenuate APLNR-mediated signaling” if, e.g., the antibody exhibitsone or more properties selected from the group consisting of: (1)inhibition of APLNR-mediated signaling in a cell-based bioassay, such asincreased accumulation of cAMP; (2) inhibition of APLNR-inducedphosphorylation of ERKs; and (3) inhibition of APLNR-mediated β-arrestininteraction, including blocking internalization.

The present invention includes antibody-fusion proteins that bind humanAPLNR and activate APLNR-mediated signaling. An antibody-fusion proteinis deemed to “activate APLNR-mediated signaling” if, e.g., the antibodyexhibits one or more properties selected from the group consisting of:(1) activation or detection of APLNR-mediated signaling in a cell-basedbioassay, such as inhibition of cAMP; (2) activation of APLNR-inducedphosphorylation of ERKs; and (3) activation of APLNR-mediated β-arrestininteraction, including internalization.

Inhibition or activation of APLNR-mediated signaling in a cell-basedbioassay means that an anti-APLNR antibody, antibody fusion protein orantigen-binding fragment thereof modifies the signal produced in cellsthat express an APLNR receptor and a reporter element that produces adetectable signal in response to APLNR binding, e.g., using the assayformats described herein, or a substantially similar assay meant tomeasure the APLNR cellular signaling. APLNR is a G protein-coupledreceptor, specifically a Gi/o-coupled receptor, whereas stimulation ofthe receptor results in inhibition of adenylate cyclase activity whichin turn effects the accumulation of cyclic AMP (cAMP) or other cellsignaling events.

For example, the present invention includes APLNR modulators thereofthat block or inhibit apelin-mediated signaling in cells expressinghuman APLNR, with an IC₅₀ of less than about 20 nM, less than about 10nM, less than about 2 nM, less than about 1 nM, less than about 900 pM,less than about 800 pM, less than about 700 pM, less than about 600 pM,less than about 500 pM, less than about 400 pM, less than about 350 pM,less than about 300 pM, less than about 250 pM, less than about 200 pM,less than about 150 pM, less than about 100 pM, less than about 90 pM,less than about 80 pM, less than about 70 pM, less than about 60 pM,less than about 50 pM, less than about 40 pM, less than about 30 pM,less than about 20 pM, or less than about 10 pM, as measured in acell-based blocking or inhibition bioassay, e.g., using the assay formatas defined in Examples 5, 8, 9 or 11 herein, or a substantially similarassay.

Inhibition of APLNR-induced phosphorylated ERK1/2 (pERK assay) intransfected cells means that an APLNR modulator inhibits or reduces theratio of pERK1/2 to total ERK in cells expressing human APLNR in thepresence of human apelin, e.g., as measured using the assay system ofExamples 6 or 10, or a substantially similar assay. For example, thepresent invention includes APLNR modulators that inhibit APLNR-mediatedratio of pERK, in the presence of apelin, with an IC₅₀ of less thanabout 50 nM, less than about 25 nM, less than about 20 nM, less thanabout 15 nM, less than about 10 nM, less than about 5 nM, less thanabout 1 nM, less than about 900 pM, less than about 800 pM, less thanabout 700 pM, less than about 600 pM, less than about 500 pM, less thanabout 400 pM or less than about 300 pM, as measured in an APLNR-inducedpERK assay, e.g., using the assay format as defined in Example 6 or 10herein, or a substantially similar assay.

In other embodiments, however, certain APLNR modulators of the presentinvention, despite having the ability to inhibit or attenuateAPLNR-mediated signaling, do not block or only partially block theinteraction of APLNR and apelin. Such antibodies, antibody-fusionproteins and antigen-binding fragments thereof, may be referred toherein as “indirect blockers.” Without being bound by theory, it isbelieved that the indirect blockers of the invention function by bindingto APLNR at an epitope that does overlap, or overlaps only partially,with the N-terminal ligand binding domain of APLNR, but nonethelessinterferes with APLNR-mediated signaling without blocking theAPLNR/apelin interaction directly.

In another embodiment of the invention, the APLNR modulator is a partialagonist or an inverse agonist. Full agonists activate the receptor to amaximal extent. Compounds having a lower effect than a full agonist arecalled partial agonists, since they stimulate signal transduction but toa lesser extent than a full agonist. Inverse agonists reduce the basallevel of the measurable or detectable signal upon binding to thereceptor, indicative of interference with or blocking endogenousactivity. In other words, some inverse agonists reduce the activity ofcertain receptors by inhibiting their constitutive activity.

In certain embodiments, the present invention includes APLNR modulatorsthereof that activate or increase signaling in cells expressing humanAPLNR, with an EC50 of less than about 100 nM, less than about 75 nM,less than about 50 nM, less than about 25 nM, less than about 10 nM,less than about 1 nM, less than about 900 pM, less than about 800 pM,less than about 700 pM, less than about 600 pM, less than about 500 pM,less than about 400 pM, less than about 350 pM, less than about 300 pM,less than about 250 pM, less than about 200 pM, less than about 150 pM,less than about 100 pM, less than about 90 pM, less than about 80 pM,less than about 70 pM, less than about 60 pM, less than about 50 pM,less than about 40 pM, less than about 30 pM, less than about 20 pM, orless than about 10 pM, as measured in a cell-based APLNR activationbioassay, e.g., using the assay format as defined in Examples 5, 8, 9 or11 herein, or a substantially similar assay.

Activation of APLNR-mediated phosphorylated ERK1/2 (pERK) in transfectedcells means that an APLNR modulator increases the ratio of pERK1/2 tototal ERK in cells expressing human APLNR, e.g., as measured using theassay system of Examples 6 or 10, or a substantially similar assay. Forexample, the present invention includes APLNR modulators that increaseAPLNR-mediated ratio of pERK, in the presence of apelin, with an EC50 ofless than about 100 nM, less than about 75 nM, less than about 50 nM,less than about 25 nM, less than about 20 nM, less than about 15 nM,less than about 10 nM, less than about 5 nM, less than about 1 nM, lessthan about 900 pM, less than about 800 pM, less than about 700 pM, lessthan about 600 pM, less than about 500 pM, less than about 400 pM orless than about 300 pM, as measured in an APLNR-induced pERK assay,e.g., using the assay format as defined in Examples 6 or 10 herein, or asubstantially similar assay.

The present invention includes APLNR modulators that bind soluble APLNRmolecules with high affinity and/or specificity. For example, thepresent invention includes antibodies and antigen-binding fragments ofantibodies that bind APLNR with a binding ratio of greater than about 20as measured by a fluorescent activated cell sorting (FACS) assay, e.g.,using the assay format as defined in Example 4 herein. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention bind APLNR with a binding ratio of greater than about 15,greater than about 20, greater than about 100, greater than about 200,greater than about 300, greater than about 400, greater than about 500,greater than about 1000, greater than about 1500, or greater than about2000, as measured by e.g., FACS, or a substantially similar assay.

The present invention also includes anti-APLNR antibodies andantigen-binding fragments thereof that specifically bind to APLNR with adissociative half-life (t %) of greater than about 10 minutes asmeasured by surface plasmon resonance at 25° C. or 37° C., e.g., usingthe well-known BIAcore™ assay format, or a substantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Receptor Assays

The cell signaling pathway of a Gi/o-coupled receptor, such as APLNR,may be measured by a variety of bioassays. Phosphorylation of ERK 1/2provides a direct physiological functional readout of activation ofGi/o-coupled GPCRs. A common method of testing for activation of aGi/o-coupled GPCR is inhibition of adenylate cyclase activity whichrequires measuring the reduction of forskolin-stimulation of cAMP levelsthat accumulate in the cell.

Activation of Gi/o-coupled receptors results in decreased adenylylcyclase activity and therefore inhibition of cAMP in the cell, via the Galpha subunits Gi or Go. To maximize the inhibition signal, forskolin (adirect activator of adenylate cyclase) is typically utilized tostimulate adenylyl cyclase in the assay, and thus cAMP, therebyrendering the inhibition signal more easily detectable. Radiometric GEHealthcare SPA™ (Piscataway, N.J., USA) and Perkin Elmer Flash-Plate™cAMP assays are available, as well as fluorescence or luminescence-basedhomogenous assays (e.g. PerkinElmer AlphaScreen™, DiscoveRx HitHunter™(Fremont, Calif., USA), and Molecular Devices FLIPR® (Sunnyvale, Calif.,USA)) to measure accumulation of intracellular cAMP.

The [³⁵S]GTPyS assay is generally useful for Gi/o-coupled receptorsbecause Gi/o is the most abundant G protein in most cells and has afaster GDP-GTP exchange rate than other G proteins (Milligan G., 2003,Trends Pharmacol Sci, 2003, 24:87-90). APLNR-mediated guanine nucleotideexchange is monitored by measuring [³⁵S]GTPyS binding to plasmamembranes prepared from cells expressing APLNR. Commercially availableScintillation Proximity Assay (SPA™) kits allow measurement of desired[³⁵S]GTPyS-bound a subunit (PerkinElmer, Waltham, Mass., USA).

The action of GPCRs that modulate cAMP levels, like APLNR, may be linkedto luciferase transcription in a cell by a cAMP response element (CRE).A CRE-luc construct (CRE-responsive luciferase) encodes a luciferasereporter gene under the control of a promoter and tandem repeats of theCRE transcriptional response element (TRE). Following activation of thereceptor, cAMP accumulation in the cell is measured by the amount ofluciferase expressed in the cell following addition of chemiluminescentdetection reagents. For APLNR, and other Gi-coupled receptors, forskolinis added to induce cAMP and a decrease in CRE activity(chemiluminescence) indicates GPCR activation. Various commercial kitsare available, such as from Promega (Madison, Wis., USA), SABiosciences(A Qiagen Company, Valencia, Calif., USA), etc.

Phosphorylated ERK (pERK) may be measured in cell lysates from cellsexpressing APLNR receptors to determine APLNR activation. Endogenousextracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2), belong toa conserved family of serine/threonine protein kinases and are involvedcellular signaling events associated with a range of stimuli. The kinaseactivity of ERK proteins is regulated by dual phosphorylation atThreonine 202/Tyrosine 204 in ERK1, and Threonine 185/Tyrosine 187 inERK2. MEK1 and MEK2 are the primary upstream kinases responsible for ERK1/2 in this pathway. Many downstream targets of ERK 1/2 have beenidentified, including other kinases, and transcription factors. In oneexample, the pERK 1/2 assay utilizes an enzyme-linked immunosorbentassay (ELISA) method to measure specific phosphorylation of ERK 1 incellular lysates of cell cultures expressing recombinant or endogenousreceptors. In another example, the pERK 1/2 assay uses a primary(non-conjugated) antibody which recognizes phosphorylated Thr202/Tyr204in ERK1 or phos-Thr185/Tyr187 in ERK2 and a secondary conjugatedantibody that recognizes the primary antibody, whereas the secondaryconjugated mAb provides a method of detection such as a conjugate reactswith an exogenously added substrate. Various commercial kits areavailable, such as AlphaScreen® SureFire™ (PerkinElmer),ThermoScientific (Waltham, Mass., USA), Sigma Aldrich (St. Louis, Mo.,USA) etc.).

In some instances, agonist binding to the receptor may initiatearrestin-mediated signaling, without triggering G protein-mediatedsignaling or slow down G protein-mediated signaling. Beta-arrestin(β-arrestin) interaction with GPCRs at the cell-surface can uncoupleheterotrimeric G proteins to the receptor and lead to other cellsignaling cascades. β-arrestin is known to trigger endocytosis andactivation of the ERK pathway. In one example assay, bioluminescenceresonance energy transfer or BRET has been used to study the interactionof GPCRs fused to Renilla luciferase (Rlu) with β-arrestin fused togreen fluorescent protein (GFP). In this example, BRET is based on thetransfer of energy between recombinant expressed GPCR-Rlu andβ-arrestin-GFP when they are in close proximity after the addition ofthe luciferase substrate coelentcrazine, thus allowing measurement ofreal-time evaluation of these protein—protein interactions in wholecells.

Other assays have been developed, such as PathHunter® GPCR assays(DiscoveRx Corp., Fremont, Calif., USA) that directly measure GPCRactivity by detecting β-arrestin interaction with the activated GPCR.Briefly, the GPCR is fused in frame with the small enzyme fragmentProLink™ and co-expressed in cells stably expressing a fusion protein ofβ-arrestin and a deletion mutant of β-galactosidase β-gal, an enzymeacceptor, or EA). Activation of the GPCR stimulates binding ofβ-arrestin to the ProLink-tagged GPCR and the complementation of the twoenzyme fragments results in formation of an active β-gal enzyme. Anincrease in enzyme activity (i.e. GPCR activation) can be measured usingchemiluminescent detection reagents.

β-arrestin molecules have been shown to regulate GPCR internalization(i.e. endocytosis) following activation of GPCRs, such as APLNR.Agonist-activation of GPCRs leads to conformational changes,phosphorylation of the receptor, and activation of β-arrestin, or otherpathways, to mediate receptor sequestration from the cell surface. Thesequestration mechanism may be a means of desensitization (i.e. receptoris degraded following internalization) or resensitization (i.e. receptoris recycled back to the cell surface). See, e.g., Claing, A., et al.2002, Progress in Neurobiology 66: 61-79, for review.

APLNR antagonists may block internalization of the receptor. APLNRagonists may induce internalization and/or resensitization of the APLNR(Lee, D K, et al. 2010, BBRC, 395:185-189). In some embodiments, theAPLNR agonist exhibits or induces increased APLNR resensitization, asmeasured by an internalization assay. In other embodiments, the APLNRagonist exhibits or induces increased cell-surface receptor copy of theAPLNR, as measured in an internalization assay. Measuring the extent(such as an increase) of receptor internalization in any internalizationassay is done by determining the difference between the noninternalizedmeasurement (i.e., cells without prior exposure to agonist) and themeasurement obtained with agonist in the assay.

Apelin receptor sequestration, and thus apelin receptor copy, may bemeasured by a number of methods well-known in the art. APLNR agoniststimulation may result in increased or decreased receptor copy on thesurface of a particular cell. For example, an apelin receptor agonistinduces APLNR internalization may have an effect of blood pressure.Receptor internalization assays are routinely done employing, forexample, fluorescently-labeled or radiolabeled ligands, orimmunofluorescent labels (fluorescently-tagged anti-receptorantibodies), followed by microscopy and digital imaging techniques (see,e.g., El Messari et al. 2004, J Neurochem, 90:1290-1301; Evans, N.,2004, Methods of Measuring Internalization of G Protein—CoupledReceptors. Current Protocols in Pharmacology. 24: 12.6.1-12.6.22).

Apelin Peptides

Apelin is produced as a prepropeptide of 77 amino acids which is cleavedto yield several shorter biologically active fragments, or apelinpeptides. As described herein, anti-APLNR antibodies may block orinterfere with the binding of apelin peptides to the APLNR.Antibody-fusion proteins comprising an apelin peptide may activate APLNRor augment APLNR activity. Any apelin peptides may be derived from thepreproapelin polypeptide (SEQ ID NO: 227) and fused to the anti-APLNRantibodies of the invention. Apelin peptides includes fragments ofapelin peptides having C-terminal deletions, some of which have beenfound to retain their cellular activities (Messari et al. 2004, JNeurochem, 90:1290-1301). Apelin peptides also include substitutedand/or modified amino acids as described herein which confer alteredactivity compared to the endogenous activity of apelin. As such, apelinanalogues may include substituted or modified amino acid(s) that removepotential cleavage sites or otherwise stabilize the protein. As such,deletion or addition of one or more C-terminal amino acids to the apelinpeptide of an apelin-Fc-fusion protein may confer increased stability,such as resistance to degradation. Such modification of apelin peptidesdoes not alter their ability to activate the APLNR. Exemplary modifiedapelin peptides are included in Tables 9 and 10A-D, e.g. SEQ ID NO: 261,SEQ ID NO: 262, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, and SEQID NO: 273. Exemplary apelin fusion proteins of the invention thatcomprise such modified apelin peptides are included in this invention.

In some embodiments, the apelin peptide comprises a fragment or analogueof the preproapelin polypeptide (SEQ ID NO: 227). An “apelin peptide” asused herein includes non-limiting exemplary apelin fragments andanalogues known in the art, e.g., an apelin peptide comprising aminoacids 6-77, 40-77, 42-77, 43-77, 47-77, 59-77, 61-77, 63-77, 64-77,65-77, 66-77, 67-77, 73-77, 1-25, 6-25, 42-64, 61-64, 61-74, 61-75,61-76, 65-76, 65-75, 66-76, 67-76, 66-75, 67-75, 42-58, 42-57, 42-56,42-55, 42-54, 42-53, or pyroglutamylated apelin65-77 ([Pyr¹]Apelin-13),of the preproapelin polypeptide (SEQ ID NO: 227). See e.g. U.S. Pat. No.6,492,324, issued on Dec. 10, 2002, and Messari et al. 2004, supra, bothof which are herein incorporated by reference.

In some embodiments, the apelin peptide is selected from the groupconsisting of apelin40-77 (apelin-38), apelin42-77 (apelin-36),apelin43-77 (apelin-35), apelin47-77 (apelin-31), apelin59-77(apelin-19), apelin61-77 (apelin-17), apelin63-77 (apelin-15),apelin64-77 (apelin-14), apelin65-77 (apelin-13), apelin66-77(apelin-12, or A12), apelin67-77 (apelin-11), apelin68-77 (apelin-10),apelin73-77 (apelin-5), apelin61-76 (apelin-K16P), apelin61-75(apelin-K15M), apelin61-74 (apelin-K14P), and [Pyr¹]Apelin-13. Certainapelin peptides are cleavage products of the preproapelin polypeptide(SEQ ID NO: 227) yielding various lengths of the C-terminus ofpreproapelin. As such, the apelin peptide consisting of amino acids42-77 of SEQ ID NO: 227 is referred to as apelin-36; the apelin peptideconsisting of amino acids 61-77 of SEQ ID NO: 227 is referred to asapelin-17; the apelin peptide consisting of amino acids 65-77 of SEQ IDNO: 227 is referred to as apelin-13; the apelin peptide consisting ofamino acids 67-77 of SEQ ID NO: 227 is referred to as apelin-11; and soon.

In some embodiments, the apelin peptide, or analogue thereof, isselected from the group consisting of apelin-36 (SEQ ID NO: 230),apelin-17 (SEQ ID NO: 229), apelin-13 (SEQ ID NO: 228) and[Pyr1]Apelin-13. In another embodiment, the apelin peptide comprisesapelin-13 (SEQ ID NO: 228), or a fragment or analogue thereof.

Further modification of apelin peptides at the C-terminus ofpreproapelin polypeptide may eliminate or interfere with enzymaticcleavage of the peptide, for example ACE2 cleavage. In some embodiments,the apelin peptide is modified to minimize degradation and to enhanceserum stability. In certain embodiments, the modified apelin peptide isselected from the group consisting of SEQ ID NO: 270 (apelin-Cter9), SEQID NO: 271 (apelin-Cter10), SEQ ID NO: 262 (apelin-Cter11), SEQ ID NO:272 (apelin-Cter11+S), SEQ ID NO: 273 (apelin-V5-11), SEQ ID NO: 269(apelin-13+5G), SEQ ID NO: 283 (apelin-13+R), SEQ ID NO: 284(apelin-13+S), and SEQ ID NO: 285 (apelin-13+H). The Apelin-antibodyfusions of the invention may be tethered to any of these modifiedpeptides, particularly at the N-terminus of the antibody heavy or lightchain(s).

Apelin peptides are rapidly cleared from the circulation and have ashort plasma half-life of no more than eight minutes (Japp, et al, 2008,J of Amer College Cardiolog, 52(11):908-13). Apelin fusion proteins ofthe invention have increased half-life compared to apelin peptides.

In other embodiments, the apelin peptide, or fragment or analoguethereof, is fused to the 5′ (N-terminal) end or the 3′ (C-terminal) endof one or both heavy chains of the antibody. In still other embodiments,the apelin peptide, or analogue thereof, is fused to the 5′ (N-terminal)end or the 3′ (C-terminal) end of one or both light chains of theantibody.

In still other embodiments, the apelin peptide, or fragment or analoguethereof, is fused to the 5′ (N-terminal) end or the 3′ (C-terminal) endof an immunoglobulin molecule, including an antigen-binding fragmentsuch as an APLNR-binding fragment, selected from the group consisting ofa Fab fragment, a F(ab′)2 fragment, an Fd fragment, an Fv fragment, ansingle-chain Fv (scFv) fragment, a dAb fragment, and an isolatedcomplementarity determining region (CDR).

Included in the invention are analogues of apelin modified to includenon-standard amino acids or modified amino acids. Such peptidescontaining non-natural, or natural but non-coded, amino acids may besynthesized by an artificially modified genetic code in which one ormode codons is assigned to encode an amino acid which is not one of thestandard amino acids. For example, the genetic code encodes 20 standardamino acids, however, three additional proteinogenic amino acids occurin nature under particular circumstances: selenocysteine, pyrrolysineand N-Formyl-methionine (Ambrogelly, et al. 2007, Nature ChemicalBiology, 3:29-35; Böck, A. et al, 1991, TIBS, 16 (12): 463-467; andThéobald-Dietrich, A., et al., 2005, Biochimie, 87(9-10):813-817).Post-translationally modified amino acids, such as carboxyglutamic acid(γ-carboxyglutamate), hydroxyproline, and hypusine, are also included.Other non-standard amino acids include, but are not limited to,citrulline, 4-benzoylphenylalanine, aminobenzoic acid, aminohexanoicacid, Nα-methylarginine, α-Amino-n-butyric acid, norvaline, norleucine,alloisoleucine, t-leucine, α-Amino-n-heptanoic acid, pipecolic acid,α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine,allothreonine, homoalanine, homoarginine, homoasparagine, homoasparticacid, homocysteine, homoglutamic acid, homoglutamine, homoisoleucine,homoleucine, homomethionine, homophenylalanine, homoserine,homotyrosine, homovaline, isonipecotic acid, β-Alanine,β-Amino-n-butyric acid, β-Aminoisobutyric acid, γ-Aminobutyric acid,α-aminoisobutyric acid, isovaline, sarcosine, naphthylalanine, nipecoticacid, N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,octahydroindole-2-carboxylic acid, penicillamine, pyroglutamic acid,sarcosine, t-butylglycine, tetrahydro-isoquinoline-3-carboxylic acid,isoserine, and α-hydroxy-γ-aminobutyric acid. A variety of formats toexpand the genetic code are known in the art and may be employed in thepractice of the invention. (See e.g. Wolfson, W., 2006, Chem Biol,13(10): 1011-12.)

Apelin analogues incorporating such non-standard amino acids orpost-translational modifications can be synthesized by known methods.Exemplary apelin analogues include Nα-methylarginine-apelin-A12analogue, [Nle75, Tyr]apelin-36, [Glp65Nle75,Tyr77]apelin-13,(Pyr1)[Met(O)11]-apelin-13, (Pyr1)-apelin-13, [d-Ala12]-A12, andN-alpha-acetyl-nona-D-arginine amide acetate.

Also included in the invention are analogues of the apelin component ofan antibody-fusion protein modified to be resistant to cleavage, forexample cleavage by angiotensin converting enzyme 2 (ACE2). Such apelinanalogues have been shown to have a marked increase in efficacy comparedto unmodified apelin ligands in in vivo models of myocardial response toischemia (Wang, et al. Jul. 1, 2013, J Am Heart Assoc. 2: e000249).

Such cleavage-protected antibody-fusion proteins comprise apelinpeptides that are modified to include substitution variants, i.e.variants made by the exchange of one amino acid for another at one ormore cleavage sites within the protein. Such amino acid substitutionsare envisioned to confer increased stability without the loss of otherfunctions or properties of the protein. Other cleavage-protectedantibody-fusion proteins comprise apelin peptides modified to includeterminal amide or acetyl groups. In some embodiments, cleavage-protectedantibody-fusion proteins comprise proteinogenic amino acids,non-standard amino acids or post-translationally modified amino acids.Some modified apelin peptides are modified to delete and/or add one ormore C-terminal amino acids without altering their ability to activatethe APLNR. Exemplary apelin fusion proteins of the invention include SEQID NO: 270 (apelin-Cter9), SEQ ID NO: 271 (apelin-Cter10), SEQ ID NO:262 (apelin-Cter11), SEQ ID NO: 272 (apelin-Cter11+S), SEQ ID NO: 269(apelin-13+5G), SEQ ID NO: 283 (apelin-13+R), SEQ ID NO: 284(apelin-13+S), and SEQ ID NO: 285 (apelin-13+H). See also, PCTInternational Publication No. WO2014/152955 A1, published on Sep. 25,2014, which is hereby incorporated by reference.

Antibody-Fusion Proteins

The present invention also provides an antibody-fusion protein orfragment thereof comprising an anti-APLNR antibody fused to an apelinpeptide sequence. Any apelin peptides or analogues described herein andknown in the art may be derived from the preproapelin polypeptide (SEQID NO: 227). Apelin peptides may be modified using ordinary molecularbiological techniques and synthetic chemistry so as to improve theirresistance to proteolytic cleavage or resistance to metal ion-relatedcleavage. Analogues of such polypeptides include substitution variantsmade by the exchange of one amino acid for another or substitution withresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

Certain non-limiting, exemplary antibody-fusion proteins of theinvention comprise heavy and light chain variable region (HCVR/LCVR)sequences selected from the group consisting of SEQ ID NO: 2/10, 18/26,34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, and 210/218; and further comprise an apelin peptidesequence, e.g. a fragment or analogue of SEQ ID NO: 227, SEQ ID NO: 228,SEQ ID NO: 229 or SEQ ID NO: 230.

In one aspect the invention, an apelin receptor (APNLR) modulatorcomprises an apelin peptide component and an Ig molecule, such as an IgGmolecule. As such, the apelin peptide component may be fused in-frame tothe N-terminus or C-terminus of the heavy chain of the Ig molecule. Theantibody-apelin fusion proteins (otherwise known as antibody-apelinfusions) may comprise a homodimer comprising two identical heavy chaindomains and an apelin peptide component fused in-frame to the N-terminusor C-terminus of one or both heavy chains of the antibody. In otherinstances, the antibody-apelin fusion protein may be a homodimercomprising two identical heavy chain domains and an apelin peptidecomponent fused in-frame to the N-terminus or C-terminus of one or bothlight chains of the antibody. In some embodiments, the Ig molecule is ananti-APLNR antibody, thus the heavy and light chain variable regions(HCVR/LCVR) are capable of binding to the APLNR. Exemplaryantibody-fusion proteins of the invention comprise heavy and light chainvariable region (HCVR/LCVR) sequences selected from the group consistingof SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, and 210/218. In otherembodiments, the antibody-apelin fusions may comprise antigen-bindingfragments including, for example, the variable regions recited hereinfused to an apelin peptide or analogue. As such, the antibody-apelinfusions comprise a Fab, F(ab′)2 or scFv fragment. A person of ordinaryskill in the art, starting with the heavy and light chain variableregion sequences disclosed herein, can easily produce variousantibody-apelin fusions which comprise one or more apelin peptidesthereof.

As with antibody molecules, antibody-apelin fusions may be monospecificor multispecific (e.g., bispecific). A multispecific antibody-apelinfusion will typically comprise at least two different variable domains,wherein one variable domain is capable of specifically binding to APLNR,and a second variable domain may capable of binding to a differentepitope on the same antigen (i.e. APLNR) or binding to a differentantigen, such as apelin. Any multispecific antibody format may beadapted for use in the context of an antibody-fusion protein of thepresent invention using routine techniques available in the art.

In some embodiments, the components or peptides of an antibody-fusionprotein are separated by a linker (or “spacer”) peptide. Such peptidelinkers are well known in the art (e.g., polyglycine) and typicallyallow for proper folding of one or both of the components of the fusionprotein. The linker provides a flexible junction region of the componentof the fusion protein, allowing the two ends of the molecule to moveindependently, and may play an important role in retaining each of thetwo moieties' appropriate functions. Therefore, the junction region actsin some cases as both a linker, which combines the two parts together,and as a spacer, which allows each of the two parts to form its ownbiological structure and not interfere with the other part. Furthermore,the junction region should create an epitope that will not be recognizedby the subject's immune system as foreign, in other words, will not beconsidered immunogenic. Linker selection may also have an effect onbinding activity of the fusion molecule. (See Huston, et al, 1988, PNAS,85:16:5879-83; Robinson & Bates, 1998, PNAS 95(11):5929-34; Arai, et al.2001, PEDS, 14(8):529-32; and Chen, X. et al., 2013, Advanced DrugDelivery Reviews 65:1357-1369.) In one embodiment, the apelin peptide isconnected to the N-terminus or to the C-terminus of the antibody-fusionpolypeptide, or fragment thereof, via one or more peptide linkers.

The length of the linker chain may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 15 or more amino acid residues, but typically is between 5and 25 residues. Examples of linkers include polyGlycine linkers, suchas Gly-Gly (2Gly), Gly-Gly-Gly (3Gly), 4Gly, 5Gly, 6Gly, 7Gly, 8Gly or9Gly. Examples of linkers also include Gly-Ser peptide linkers such asSer-Gly (SG), Gly-Ser (GS), Gly-Gly-Ser (G2S), Ser-Gly-Gly (SG2), G3S,SG3, G4S, SG4, G5S, SG5, G6S, SG6, (G4S)n, (S4G)n, wherein n=1 to 10.(Gly-Gly-Gly-Gly-Ser)3 is also known as (G4S)3 (SEQ ID NO: 233), whereinn=3 indicating that the particular sequence is repeated 3 times. Any oneof the linkers described herein may be repeated to lengthen the linkeras needed.

In one such embodiment of the invention, the apelin peptide is connectedto the N-terminus or to the C-terminus of the antibody-fusion protein,or fragment thereof, via one or more Gly-Ser peptide linkers.

In some embodiments, the peptide linker is selected from the groupconsisting of (Gly-Gly-Gly-Gly-Ser)1 (SEQ ID NO: 231),(Gly-Gly-Gly-Gly-Ser)2 (SEQ ID NO: 232), and (Gly-Gly-Gly-Gly-Ser)3 (SEQID NO: 233).

In other embodiments, a signal peptide is encoded upstream of theantibody-fusion protein in an expression vector. In certain embodiments,a linker or spacer is fused in-frame between the C-terminus of thesignal peptide and N-terminus of the antibody-fusion protein. Suchsignal peptides are known in the art and may be employed to direct thepolypeptide into a cell's secretory pathway.

Exemplary apelin fusion proteins of the invention are more stable thanapelin peptides alone. Some apelin fusion proteins of the invention areresistant to enzymatic degradation. Exemplary antibody-fusion proteinsof the invention comprise heavy and light chain variable region(HCVR/LCVR) sequences fused to apelin peptides and optionally fused to alinker, and are selected from the group consisting of SEQ ID NO:130/235, 130/237, 239/138, 241/138, 243/138, 245/138, 247/122, 114/249,114/251, 253/26, 255/26, 257/26, 259/26, 274/138, 275/138, 276/138,277/138, 278/138, 279/26, 280/26, 281/26, and 282/26.

Epitope Mapping and Related Technologies

The present invention includes anti-APLNR antibodies and antibody-fusionproteins which interact with one or more amino acids of APLNR. Forexample, the present invention includes anti-APLNR antibodies thatinteract with one or more amino acids located within an extracellular ortransmembrane domain of APLNR. The epitope to which the antibodies bindmay consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) aminoacids of APLNR. Alternatively, the epitope may consist of a plurality ofnon-contiguous amino acids (or amino acid sequences) of APLNR.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-APLNR antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. H1M9207N, H2aM9209N, H2aM9222N, H2aM9227N,H2aM9227N, H2aM9228N, H2aM9230N, H2aM9232N, H4H9092P, H4H9093P,H4H9101P, H4H9103P, H4H9104P, H4H9112P, H4H9113P, etc.). Likewise, thepresent invention also includes anti-APLNR antibodies that compete forbinding to APLNR with any of the specific exemplary antibodies describedherein (e.g. H1M9207N, H2aM9209N, H2aM9222N, H2aM9227N, H2aM9227N,H2aM9228N, H2aM9230N, H2aM9232N, H4H9092P, H4H9093P, H4H9101P, H4H9103P,H4H9104P, H4H9112P, H4H9113P, etc.).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-APLNR antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-APLNR antibody of the invention, the reference antibodyis allowed to bind to an APLNR protein. Next, the ability of a testantibody to bind to the APLNR molecule is assessed. If the test antibodyis able to bind to APLNR following saturation binding with the referenceanti-APLNR antibody, it can be concluded that the test antibody binds toa different epitope than the reference anti-APLNR antibody. On the otherhand, if the test antibody is not able to bind to the APLNR moleculefollowing saturation binding with the reference anti-APLNR antibody,then the test antibody may bind to the same epitope as the epitope boundby the reference anti-APLNR antibody of the invention. Additionalroutine experimentation (e.g., peptide mutation and binding analyses)can then be carried out to confirm whether the observed lack of bindingof the test antibody is in fact due to binding to the same epitope asthe reference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, BIAcore™, flow cytometry or any otherquantitative or qualitative antibody-binding assay available in the art.In accordance with certain embodiments of the present invention, twoantibodies bind to the same (or overlapping) epitope if, e.g., a 1-, 5-,10-, 20- or 100-fold excess of one antibody inhibits binding of theother by at least 50% but preferably 75%, 90% or even 99% as measured ina competitive binding assay (see, e.g., Junghans et al., 1990, CancerRes. 50:1495-1502). Alternatively, two antibodies are deemed to bind tothe same epitope if essentially all amino acid mutations in the antigenthat reduce or eliminate binding of one antibody reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-APLNR antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to an APLNRprotein under saturating conditions followed by assessment of binding ofthe test antibody to the APLNR molecule. In a second orientation, thetest antibody is allowed to bind to an APLNR molecule under saturatingconditions followed by assessment of binding of the reference antibodyto the APLNR molecule. If, in both orientations, only the first(saturating) antibody is capable of binding to the APLNR molecule, thenit is concluded that the test antibody and the reference antibodycompete for binding to APLNR. As will be appreciated by a person ofordinary skill in the art, an antibody that competes for binding with areference antibody may not necessarily bind to the same epitope as thereference antibody, but may sterically block binding of the referenceantibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human APLNR.

Using VELOCIMMUNE™ technology, for example, or any other known methodfor generating fully human monoclonal antibodies, high affinity chimericantibodies to APLNR are initially isolated having a human variableregion and a mouse constant region. As in the experimental sectionbelow, the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Ifnecessary, mouse constant regions are replaced with a desired humanconstant region, for example wild-type or modified IgG1, IgG2 or IgG4,to generate a fully human anti-APLNR antibody. While the constant regionselected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region. In certain instances, fully human anti-APLNR antibodiesare isolated directly from antigen-positive B cells.

Bioequivalents

The anti-APLNR antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman APLNR. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-APLNR antibody-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-APLNR antibody or antibody fragment that isessentially bioequivalent to an anti-APLNR antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-APLNR antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-APLNR antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-APLNR antibodies that bind to human APLNR but not to APLNR fromother species. The present invention also includes anti-APLNR antibodiesthat bind to human APLNR and to APLNR from one or more non-humanspecies. For example, the anti-APLNR antibodies of the invention maybind to human APLNR and may bind or not bind, as the case may be, to oneor more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog,rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus orchimpanzee APLNR. According to certain exemplary embodiments of thepresent invention, anti-APLNR antibodies are provided which specificallybind human APLNR (SEQ ID NO: 225) and cynomolgus monkey (e.g., Macacafascicularis) APLNR (SEQ ID NO: 226).

Immunoconjugates

The invention encompasses anti-APLNR monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-APLNR antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human APLNR or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention. See, e.g., U.S. Application Publication No.US20100331527A1, published Dec. 30, 2010, which is herein incorporate byreference.

In some aspects, the antibody, antibody-fusion protein orantigen-binding fragment is a bispecific antibody wherein eachantigen-binding fragment of such molecule or antibody comprises a HCVRpaired with a LCVR region. In certain embodiments, the bispecificantibody comprises a first antigen-binding fragment and a second antigenbinding fragment each comprising different, distinct HCVRs paired with aLCVR region. In some embodiments, the bispecific antibodies areconstructed comprising a first antigen-binding fragment thatspecifically binds a first antigen, wherein the first antigen-bindingfragment comprises an HCVR/LCVR pair derived from a first antibodydirected against the first antigen, and a second antigen-bindingfragment that specifically binds a second antigen, wherein the secondantigen-binding fragment comprises an HCVR derived from a secondantibody directed against a second antigen paired with an LCVR derivedfrom the first antibody (e.g., the same LCVR that is included in theantigen-binding fragment of the first antibody). In some embodiments,the heavy chain of at least one of the antibodies, i.e. the firstantibody or the second antibody or both antibodies, in a bispecificantibody comprises a modified heavy chain constant region

In some aspects of the invention, two antibodies, or two heavy chains,having different specificity use the same light chain in a bispecificantibody. In some embodiments, at least one of the heavy chains ismodified in the CH3 domain resulting in a differential affinity betweeneach heavy chain of the bispecific antibody and an affinity reagent,such as Protein A, for ease of isolation. In another embodiment, atleast one of the heavy chains in such bispecific antibody comprises anamino acid modification at i) 95R or ii) 95R and 96F in the IMGTnumbering system (95R and 96F correspond to 435R and 436F in the EUnumbering system).

In still other aspects, the antibody is a bispecific antibody whereinthe bispecific antibody comprises: (a) a first heavy chain comprising anantigen-binding fragment capable of recognizing and binding to a firsttarget antigen, (b) a second heavy chain comprising an antigen-bindingfragment capable of recognizing and binding to a second target antigen,(c) a common light chain antigen-binding fragment capable of recognizingand binding to the first or second target antigen. In another aspect, atleast one of the heavy chains of (a) or (b) in such bispecific antibodyhereinabove comprises an amino acid modification (i) 95R or (ii) 95R and96F in the IMGT numbering system [(i) 435R or (ii) 435R and 436F (EUnumbering)].

Exemplary bispecific formats may be used in the context of the inventioncomprising any of the HCVR and/or LCVR sequences described herein. Insome embodiments, the first antigen is a first epitope on hAPLNR, andthe second antigen is a second epitope on hAPLNR. In other embodiments,the first antigen is APLNR and the second antigen is apelin. In certainembodiments, the first antibody comprises an anti-APLNR antigen-bindingfragment described herein. In other embodiments, the second antibodycomprises an anti-apelin antigen-binding fragment. Such anti-apelinantigen-binding fragments are known in the art (see, e.g. PCTInternational Publication No. WO2013/012855, published Jan. 24, 2013,which is herein incorporated by reference).

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mabe bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al. 2013, J. Am. Chem. Soc. 9; 135(1):340-6 [Epub:Dec. 21, 2012]).

Further exemplary multispecific formats can be used in the context ofthe present invention include, without limitation, e.g., involving afirst antigen-binding domain that specifically binds a target molecule,and a second antigen-binding domain that specifically binds aninternalizing effector protein, wherein such second antigen-bindingdomains are capable of activating and internalizing the APLNR. (See U.S.Application Publication No. 2013/0243775A1, published on Sep. 19, 2013,which is incorporated by reference.)

pH-Dependent Binding

The present invention provides antibodies, antibody-fusion proteins andantigen-binding fragments thereof that bind APLNR in a pH-dependentmanner. For example, an anti-APLNR antibody of the invention may exhibitreduced binding to APLNR at acidic pH as compared to neutral pH.Alternatively, an anti-APLNR antibody of the invention may exhibitenhanced binding to its antigen at acidic pH as compared to neutral pH.

In certain instances, “reduced binding to APLNR at acidic pH as comparedto neutral pH” is expressed in terms of a binding quotient of thebinding ratio of the antibody to cells expressing APLNR at acidic pH tothe binding ratio of the antibody to cells expressing APLNR at neutralpH (or vice versa). For example, an antibody or antigen-binding fragmentthereof may be regarded as exhibiting “reduced binding to APLNR atacidic pH as compared to neutral pH” for purposes of the presentinvention if the antibody or antigen-binding fragment thereof exhibitsan acidic/neutral binding quotient of about 3.0 or greater. In certainembodiments, the acidic/neutral binding quotient for an antibody orantigen-binding fragment of the present invention can be about 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0,40.0, 50.0, 60.0, 70.0, 100.0 or greater.

Alternatively, “reduced binding to APLNR at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of theantibody binding to APLNR at acidic pH to the K_(D) value of theantibody binding to APLNR at neutral pH (or vice versa). The term“K_(D)” (M), as used herein, refers to the dissociation equilibriumconstant of a particular ligand-receptor interaction. There is aninverse relationship between K_(D) and binding affinity, therefore thesmaller the K_(D) value, the higher the affinity. Thus, the term “loweraffinity” relates to a lower ability to form an interaction andtherefore a larger K_(D) value. For example, an antibody orantigen-binding fragment thereof may be regarded as exhibiting “reducedbinding to APLNR at acidic pH as compared to neutral pH” for purposes ofthe present invention if the antibody or antigen-binding fragmentthereof exhibits an acidic/neutral K_(D) ratio of about 3.0 or greater.In certain embodiments, the acidic/neutral K_(D) ratio for an antibodyor antigen-binding fragment of the present invention can be about 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0,30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained. As used herein, the expression “acidic pH” means a pH of about6.0 or less, about 5.5 or less, or about 5.0 or less. The expression“acidic pH” includes pH values of about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75,5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1,5.05, 5.0, or less. As used herein, the expression “neutral pH” means apH of about 7.0 to about 7.4. The expression “neutral pH” includes pHvalues of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-APLNR antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations”PDA, 1998, J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with APLNR activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-APLNRantibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

Experiments using mouse model systems, such as conducted by the presentinventors, have contributed to the identification of various diseasesand conditions that can be treated, prevented and/or ameliorated byAPLNR antagonism. For example, Apelin(−/−) knockout mice exhibit anobvious impairment of normal developmental angiogenesis in the eye. Inaddition, APLNR(−/−) mice have altered fluid homeostasis and an alteredresponse to osmotic stress (Roberts, Em et al. 2009, J Endocrinol202:453-462; Roberts, E M, et al. 2010, J Endocrinol 22:301-308). Inanother example, APLNR^(−/−) mice showed normal baseline blood pressureand heart rate, but lack the hypotensive response to apelin (Charo, etal. 2009, Am J Physiol Heart Circ Physiol 297: H1904-H1913 [Epub on Sep.18, 2009]). Furthermore, exemplary anti-APLNR antibodies are antagonistsof the receptor and exhibit an APLNR-mediated anti-angiogenic effect inthe eye vasculature as measured in a retinal vascular development (RVD)model.

Antagonists of the receptor, such as the functional antagonist derivedby modifying apelin-13 at its C-terminal phenylalanine (F) to alanine(A) (i.e. apelin-13(F13A)), were shown to block the hypotensive actionof the APLNR (Lee, et al. Endocrinol 2005, 146(1):231-236).

Apelin peptide may promote obesity through adipose tissue expansion.Apelin is induced by hypoxia and drives angiogenesis within the hypoxicinterior of expanding adipose tissue. (Kunduzova O, et al., 2008, FASEBJ, 22:4146-4153). Anti-APLNR antibodies act as inhibiting agents of thismechanism, in a tissue-specific manner, and can promote weight loss ortreat obesity. Therefore, Anti-APLNR antibodies may be administered totreat obesity and to promote weight loss.

Pathological angiogenesis, involved in promoting tumor growth orneovascularization in the retina may be responsive to apelin or APLNRantagonist. (Kojima, Y. and Quertermous, T., 2008, Arterioscler ThrombVasc Biol, 28:1687-1688; Rayalam, S. et al. 2011, Recent Pat AnticancerDrug Discov 6(3):367-72). As such, anti-APLNR antibodies may beadministered to slow tumor growth or metastasis, or to treat cancer andmetastatic disease.

Apelin may associate with a progressive overexpression of VEGF and GFAP,suggesting a role for apelin-mediated signaling in the progression ofdiabetic retinopathy (DR) to a proliferative phase. Anti-APLNRantibodies may be administered for early prevention and treatment of DR(Lu, Q. et al, 2013, PLoS One 8(7):e69703).

APLNR antagonists may also reduce angiogenesis and improve function,such as in fibrotic tissues, by ameliorating the effects of anoveractive apelin system caused by a pathogenic disease (Principe, etal., 2008, Hepatology, 48(4):1193-1201; Reichenbach, et al., 2012, JPET340(3):629-637). Without being bound by any one theory, blocking theapelin system may slow the formation of excess fibrous connective tissuein an organ or tissue in a reparative or reactive process, such as in apathological condition like cirrhosis. As such, anti-APLNR antibodiesmay be used as inhibiting agents administered to slow or prevent theprogression of fibrosis, or to treat fibrosis.

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by APLNR expression, signaling, or activity,or treatable by blocking the interaction between APLNR and a APLNRligand (e.g., apelin) or otherwise inhibiting APLNR activity and/orsignaling. For example, the present invention provides methods fortreating a disease or disorder selected from the group consisting ofobesity, cancer, metastatic disease, retinopathy, fibrosis, andpathological angiogenesis. In one embodiment, the APLNR modulatorpromotes weight loss. In another embodiment, the APLNR modulatordecreases pathological angiogenesis or neovascularization. In otherembodiments, the APLNR modulator decreases or inhibits tumor growth.

In other circumstances, including experiments using animal modelsystems, treatment of various diseases and conditions has been showneffective by APLNR agonism or partial agonism. Agonists of APLNR, suchas apelin, have been administered for the management of cardiovascularconditions, such as inotropic agents, specifically positive inotropicagents. Without being bound to a particular theory, positive inotropicagents increase myocardial contractility, and are used to supportcardiac function in conditions such as congestive heart failure,myocardial infarction, cardiomyopathy, and others. (See Dai, et al.,2006, Eur J Pharmacol 553(1-3): 222-228; Maguire, et al, Hypertension.2009; 54:598-604; and Berry, M., et al., 2004 Circulation,110:11187-11193.) Apelin-induced vasodilation may be protective inischemia-reperfusion injury. Promotion of angiogenesis and induction oflarger nonleaky vessels by apelin peptides may contribute to functionalrecovery from ischemia. (Eyries M, et al., 2008, Circ Res 103:432-440;Kidoya H, et al., 2010, Blood 115:3166-3174).

Apelin receptor agonists are considered pro-angiogenic agents which areadministered to increase cardiac output, improve cardiac function,stabilize cardiac function, limit a decrease in cardiac function, orpromote new blood vessel growth in an ischemic or damaged area of theheart or other tissue. Thus, agonistic APLNR modulators of the inventionare useful to promote angiogenesis and therefore treat ischemia, restorebloodflow to ischemic organs and tissues, for example to treat limbischemia, peripheral ischemia, renal ischemia, ocular ischemia, cerebralischemia, or any ischemic disease.

Apelin has also been shown to improve glucose tolerance and enhanceglucose utilization, by muscle tissue, in obese insulin-resistant mice(Dray et al., 2008, Cell Metab 8:437-445). Apelin KO mice havediminished insulin sensitivity (Yue at al., 2010, Am J PhysiolEndocrinol Metab 298:E59-E67). As such, agonistic Antibody-Apelin fusionproteins may improve glucose-tolerance in the treatment ofinsulin-resistant diabetes, and thus may be administered for themanagement of metabolic conditions related to diabetes.

Changes in muscle apelin mRNA levels are also correlative withwhole-body insulin sensitivity improvements (Besse-Patin, A. et al.,2013 Aug. 27, Int J Obes (Lond). doi: 10.1038/ijo.2013.158, [Epub aheadof print]). Due to such metabolic improvements in muscle tissue, andapelin-induced vasodilation, agonistic Antibody-Apelin fusion proteinsmay also be administered to stimulate muscle growth and endurance.

It has been shown that primary HIV-1 isolates can also use APLNR as acoreceptor and synthetic apelin peptides inhibited HIV-1 entry intoCD4-APLNR-expressing cells (Cayabyab, M., et al., 2000, J. Virol., 74:11972-11976). Agonistic Antibody-Apelin fusion proteins may also treatHIV infection. Apelin-neuroprotection is also seen where apelin peptidesact through signaling pathways to promote neuronal survival (Cheng, B,et al., 2012, Peptides, 37(1):171-3). Thus, Antibody-Apelin fusionproteins may promote or increase survival of neurons, or treat neuronalinjury or neurodegeneration. Apelin receptor agonists have beendescribed as hot flash suppressants. (See WO2012/133825, published Oct.4, 2012), therefore Antibody-Apelin fusion proteins of the invention mayalso be administered to treat, improve or suppress hot flash symptoms ina subject.

The antibody-fusion proteins of the invention are useful, inter alia,for the treatment, prevention and/or amelioration of any disease ordisorder associated with or mediated by activating or stimulating APLNRexpression, signaling, or activity. For example, the present inventionprovides methods for treating a disease or disorder selected from thegroup consisting of cardiovascular disease, acute decompensated heartfailure, congestive heart failure, myocardial infarction,cardiomyopathy, ischemia, ischemia/reperfusion injury, pulmonaryhypertension, diabetes, neuronal injury, neurodegeneration, hot flashsymptoms, fluid homeostasis, and HIV infection. In some embodiments, theAPLNR modulator is useful to treat or alleviate ischemia and reperfusioninjury, such as to limit ischemia/reperfusion (I/R) injury or delay theonset of necrosis of the heart tissue, or to provide preventivetreatment, for example, to protect the heart from ischemia/reperfusion(I/R) injury, improve cardiac function, or limit the developmentmyocardial infarction.

In the context of the methods of treatment described herein, the APLNRmodulator may be administered as a monotherapy (i.e., as the onlytherapeutic agent) or in combination with one or more additionaltherapeutic agents (examples of which are described elsewhere herein).

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the APLNR modulators described herein in combinationwith one or more additional therapeutically active components, andmethods of treatment comprising administering such combinations tosubjects in need thereof.

The APLNR modulators of the present invention may be co-formulated withand/or administered in combination with, e.g., VEGF inhibitors,including small-molecule angiogenic inhibitors, and antibodies that bindto cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9,IL-11, IL-12, IL-13, IL-17, IL-18, IL-21, IL-23, IL-26, or antagonistsof their respective receptors. Other additional therapeutically activecomponents may include blood pressure medication, calcium channelblockers, digitalis, anti-arrhythmics, ACE inhibitors, anti-coagulants,immunosuppressants, pain relievers, vasodilators, etc.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan APLNR modulator of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an APLNR modulator “in combination with” an additionaltherapeutically active component). The present invention includespharmaceutical compositions in which an APLNR modulator of the presentinvention is co-formulated with one or more of the additionaltherapeutically active component(s) as described elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an APLNR modulator (or a pharmaceutical composition comprisinga combination of an APLNR modulator and any of the additionaltherapeutically active agents mentioned herein) may be administered to asubject over a defined time course. The methods according to this aspectof the invention comprise sequentially administering to a subjectmultiple doses of an APLNR modulator of the invention. As used herein,“sequentially administering” means that each dose of APLNR modulator isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of anAPLNR modulator, followed by one or more secondary doses of the APLNRmodulator, and optionally followed by one or more tertiary doses of theAPLNR modulator.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the APLNR modulator of theinvention. Thus, the “initial dose” is the dose which is administered atthe beginning of the treatment regimen (also referred to as the“baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of APLNRmodulator, but generally may differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof APLNR modulator contained in the initial, secondary and/or tertiarydoses varies from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, two or more(e.g., 2, 3, 4, or 5) doses are administered at the beginning of thetreatment regimen as “loading doses” followed by subsequent doses thatare administered on a less frequent basis (e.g., “maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1%, 2,2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11%,12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%,20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of APLNR modulator which is administered to apatient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an APLNR modulator. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.).

Diagnostic Uses of the Antibodies

The anti-APLNR antibodies of the present invention may also be used todetect and/or measure APLNR, or APLNR-expressing cells in a sample,e.g., for diagnostic purposes. For example, an anti-APLNR antibody, orfragment thereof, may be used to diagnose a condition or diseasecharacterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of APLNR. Exemplarydiagnostic assays for APLNR may comprise, e.g., contacting a sample,obtained from a patient, with an anti-APLNR antibody of the invention,wherein the anti-APLNR antibody is labeled with a detectable label orreporter molecule. Antibody-fusion proteins of the invention may beemployed in such an assay, wherein the apelin fusion component or theantibody component is labeled with a detectable label or reportermolecule. Alternatively, an unlabeled anti-APLNR antibody can be used indiagnostic applications in combination with a secondary antibody whichis itself detectably labeled. The detectable label or reporter moleculecan be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescentor chemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure APLNR in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in APLNR diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of APLNR protein, orfragments thereof, under normal or pathological conditions. Generally,levels of APLNR in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal APLNR levels or activity) will be measured to initiallyestablish a baseline, or standard, level of APLNR. This baseline levelof APLNR can then be compared against the levels of APLNR measured insamples obtained from individuals suspected of having an APLNR relateddisease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to Human APLNR

An immunogen comprising human APLNR was administered directly, with anadjuvant to stimulate the immune response, to a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The antibody immune response was monitored by ananti-APLNR immunoassay. When a desired immune response was achievedsplenocytes were harvested and fused with mouse myeloma cells topreserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce anti-APLNR antibodies. Using this technique several anti-APLNRchimeric antibodies (i.e., antibodies possessing human variable domainsand mouse constant domains) were obtained; exemplary antibodiesgenerated in this manner were designated as follows: H1M9207N,H2aM9230N, and H2aM9232N. The human variable domains from the chimericantibodies were subsequently cloned onto human constant domains to makefully human anti-APLNR antibodies as described herein.

Anti-APLNR antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in US PatentApplication Publication No. 200710280945A1, published on Dec. 6, 2007.Using this method, several fully human anti-APLNR antibodies (i.e.,antibodies possessing human variable domains and human constant domains)were obtained; exemplary antibodies generated in this manner weredesignated as follows: H4H9092P, H4H9093P, H4H9101P, H4H9103P, H4H9104P,H4H9112P, and H4H9113P.

Certain biological properties of the exemplary anti-APLNR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs, and CDR sequences, of selected anti-APLNR antibodies andtheir corresponding antibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 H1M9207N 2 4 6 8 10 12 14 16 H2aM9209N 18 20 22 24 2628 30 32 H2aM9222N 34 36 38 40 42 44 46 48 H2aM9227N 50 52 54 56 58 6062 64 H2aM9228N 66 68 70 72 74 76 78 80 H2aM9230N 82 84 86 88 90 92 9496 H2aM9232N 98 100 102 104 106 108 110 112 H4H9092P 114 116 118 120 122124 126 128 H4H9093P 130 132 134 136 138 140 142 144 H4H9101P 146 148150 152 154 156 158 160 H4H9103P 162 164 166 168 170 172 174 176H4H9104P 178 180 182 184 186 188 190 192 H4H9112P 194 196 198 200 202204 206 208 H4H9113P 210 212 214 216 218 220 222 224

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” or “H4H”), followed by a numericalidentifier (e.g. “9207,” “9209,” or “9230” as shown in Table 1),followed by a “P,” or “N” suffix. Thus, according to this nomenclature,an antibody may be referred to herein as, e.g., “H1M9207N,” “H2aM9209N,”“H4H9113P,” etc. The H1M, H2aM, and H4H prefixes on the antibodydesignations used herein indicate the particular Fc region isotype ofthe antibody. For example, an “I-11M” antibody has a mouse IgG1 Fc, and“H2aM” antibody has a mouse IgG2a Fc, whereas an “H4H” antibody has ahuman IgG4 Fc. As will be appreciated by a person of ordinary skill inthe art, an antibody having a particular Fc isotype can be converted toan antibody with a different Fc isotype (e.g., an antibody with a mouseIgG1 Fc can be converted to an antibody with a human IgG4, etc.), but inany event, the variable domains (including the CDRs)—which are indicatedby the numerical identifiers shown in Table 1—will remain the same, andthe binding properties are expected to be identical or substantiallysimilar regardless of the nature of the Fc domain. In one example, theantibody designated H2aM9209N was engineered to have a human IgG4 Fcdomain. Thus, the antibody designated herein as H4H9209N has a humanIgG4 domain and has the same heavy chains (HC) or light chains (LC), andthus substantially the same binding and cellular activitycharacteristics as antibody H2aM9209N.

Example 3. Generation of Antibody-Fusion Proteins

To manufacture the nucleic acid encoding the antibody fusion proteins ofthe invention, the heavy and light chain variable region amino acidsequence pairs, and CDR sequences, of selected anti-APLNR antibodieswere amplified via polymerase chain reaction and either the heavy chain(HC) or light chain (LC) was ligated to sequence encoding apelin-13 (SEQID NO: 228), or to modified apelin peptides, such as the C-terminaltruncated apelin-Cter9 (SEQ ID NO: 270), apelin-Cter10 (SEQ ID NO: 271),or apelin-Cter11 (SEQ ID NO: 262). Contiguous nucleic acid sequencesthat encode the apelin-containing antibody-fusion proteins of Table 2Aand Table 2B were cloned into expression vectors using standard PCR andrestriction endonuclease cloning techniques.

Table 2A identifies heavy and light chain variable region amino acidsequence pairs, heavy chain Fc regions, and apelin fusion pattern ofselected antibody-fusion proteins and their correspondingantibody-fusion nomenclature. In some exemplified antibody-fusionproteins, the apelin peptide is fused to the heavy chain variable region(HCVR), and in other examples, the apelin peptide is fused to the lightchain variable region (LCVR) or the light chain (which may or may notinclude a light chain constant region). In some examples, the apelinpeptide is fused to the polypeptide via a linker. Table 2B indicatescertain exemplified sequence pairs, for example either the heavy orlight chain sequence is fused to an apelin sequence (fusion).

TABLE 2A HCVR HC LCVR Apelin-13 Antibody- Fusion SEQ ID constant SEQ ID(SEQ ID NO: 228) Designation NO: region NO: fused to: LC or HCH4H9093P-1-NVK3 130 human IgG4 138 N-terminal end LC with Fc (G4S)3linker H4H9093P-2-CVK3 130 human IgG4 138 C-terminal end LC with Fc(G4S)3 linker H4H9093P-3-NVH3 130 human IgG4 138 N-terminal end HC withFc (G4S)3 linker H4H9093P-4-NVH0 130 human IgG4 138 N-terminal end HCwith no Fc linker H4H9093P-5-NVH1 130 human IgG4 138 N-terminal end HCwith G4S Fc linker H4H9093P-6-NVH2 130 human IgG4 138 N-terminal end HCwith Fc (G4S)2 linker H4H9092P-1-NVH3 114 human IgG4 122 N-terminal endHC with Fc (G4S)3 linker H4H9092P-2-NVK3 114 human IgG4 122 N-terminalend LC with Fc (G4S)3 linker H4H9092P-3-CVK3 114 human IgG4 122C-terminal end LC with Fc (G4S)3 linker H4H9209N-1-NVH0 18 human IgG4 26N-terminal end HC with no Fc linker H4H9209N-2- NVH1 18 human IgG4 26N-terminal end LC with G4S Fc linker H4H9209N-3- NVH2 18 human IgG4 26N-terminal end HC with Fc (G4S)2 linker H4H9209N-4-NVH3 18 human IgG4 26N-terminal end HC with Fc (G4S)3 linker HCVR HC LCVR Antibody- FusionSEQ ID constant SEQ ID Modified Apelin Designation NO: region NO: fusedto HC H4H9093P-APN9- 130 human IgG4 138 Apelin-Cter9 (SEQ ID NO: 270)(G4S)3 Fc N-terminal end HC fusion with (G4S)3 linker H4H9093P-APN10-130 human IgG4 138 Apelin-Cter10 (SEQ ID (G4S)3 Fc NO: 271) N-terminalend HC fusion with (G4S)3 linker H4H9093P-APN11- 130 human IgG4 138Apelin-Cter11 (SEQ ID (G4S)3 Fc NO: 262) N-terminal end HC fusion with(G4S)3 linker H4H9093P- 130 human IgG4 138 Apelin-Cter11 + S (SEQ IDAPN11 + S -(G4S)3 Fc NO: 272) N-terminal end HC fusion with (G4S)3linker H4H9093P-APNV5- 130 human IgG4 138 Apelin-V5linker-Cter11 (SEQ11-(G4S)3 Fc ID NO: 273) N-terminal end HC fusion with (G4S)3 linkerH4H9209N-APN9- 18 human IgG4 26 Apelin-Cter9 (SEQ ID NO: 270) (G4S)3 FcN-terminal end HC fusion with (G4S)3 linker H4H9209N-APN10- 18 humanIgG4 26 Apelin-Cter10 (SEQ ID (G4S)3 Fc NO: 271) N-terminal end HCfusion with (G4S)3 linker H4H9209N-APN11- 18 human IgG4 26 Apelin-Cter11(SEQ ID (G4S)3 Fc NO: 262) N-terminal end HC fusion with (G4S)3 linkerH4H9209N- 18 human IgG4 26 Apelin-Cter11 + S (SEQ ID APN11 + S -(G4S)3Fc NO: 272) N-terminal end HC fusion with (G4S)3 linker

TABLE 2B Amino acid sequence pairs LC fusion, HCVR fusion LCVR fusionAntibody- Fusion or HCVR or LCVR Designation SEQ ID NO: SEQ ID NO:H4H9093P-1-NVK3 130 (HCVR) 235 (LCVR fusion) H4H9093P-2-CVK3 130 (HCVR)237 (LC fusion) H4H9093P-3-NVH3 239 (HCVR fusion) 138 (LCVR)H4H9093P-4-NVH0 241 (HCVR fusion) 138 (LCVR) H4H9093P-5-NVH1 243 (HCVRfusion) 138 (LCVR) H4H9093P-6-NVH2 245 (HCVR fusion) 138 (LCVR)H4H9092P-1-NVH3 247 (HCVR fusion) 122 (LCVR) H4H9092P-2-NVK3 114 (HCVR)249 (LCVR fusion) H4H9092P-3-CVK3 114 (HCVR) 251 (LC fusion)H4H9209N-1-NVH0 253 (HCVR fusion)  26 (LCVR) H4H9209N-2- NVH1 255 (HCVRfusion)  26 (LCVR) H4H9209N-3- NVH2 257 (HCVR fusion)  26 (LCVR)H4H9209N-4- NVH3 259 (HCVR fusion)  26 (LCVR) H4H9093P-APN9- 274 (HCVRfusion) 138 (LCVR) (G4S)3 H4H9093P-APN10- 275 (HCVR fusion) 138 (LCVR)(G4S)3 H4H9093P-APN11- 276 (HCVR fusion) 138 (LCVR) (G4S)3 H4H9093P- 277(HCVR fusion) 138 (LCVR) APN11 + S -(G4S)3 H4H9093P-APNV5- 278 (HCVRfusion) 138 (LCVR) 11-(G4S)3 H4H9209N-APN9- 279 (HCVR fusion)  26 (LCVR)(G4S)3 H4H9209N-APN10- 280 (HCVR fusion)  26 (LCVR) (G4S)3H4H9209N-APN11- 281 (HCVR fusion)  26 (LCVR) (G4S)3 H4H9209N- 282 (HCVRfusion)  26 (LCVR) APN11 + S-(G4S)3

Certain biological properties of the exemplary antibody-fusion proteinsgenerated in accordance with these methods are described in detail inthe Examples set forth below.

Example 4. Antibody and Antibody-Fusion Protein Binding to Human APLNRas Determined by FACS Analysis

Binding ratios for human APLNR binding to purified anti-APLNR monoclonalantibodies were determined by a fluorescence-activated cell sorting(FACS) binding assay. HEK293 cell lines stably expressing thefull-length human APLNR (hAPLNR; SEQ ID NO: 225) or the full lengthcynomolgus APLNR (MfAPLNR; SEQ ID NO: 226) along with a luciferasereporter [cAMP response element (CRE,4X)-luciferase-IRES-GFP] weregenerated by well-known methods. The resulting stable cell lines,HEK293/CRE-luc/hAPLNR and HEK293/CRE-luc/MfAPLNR, were maintained inDMEM containing 10% FBS, NEAA, and penicillin/streptomycin with either100 μg/mL Hygromycin B for hAPLNR cells or 500 μg/mL G418 for MfAPLNRcells.

For the FACS analysis, HEK293 parental, HEK293/CRE-luc/hAPLNR, and,HEK293/CRE-luc/mfAPLNR cells were dissociated using Enzyme FreeDissociation reagent (# S-004, Millipore, Billerica, Mass., USA) and 10⁶cells/well were plated onto 96-well v-bottom plates in PBS containing 1%FBS. The cells were then incubated with 10 μg/mL of anti-APLNRantibodies or negative isotype control antibodies for 30 minutes at 4°C., followed by washing with PBS containing 1% FBS and incubation with 4μg/mL of either anti-mouse IgG antibody conjugated with Alexa 647 (#115-607-003, Jackson ImmunoResearch, West Grove, Pa., USA) or anti-humanIgG antibody conjugated with Alexa 488 (# 109-547-003, JacksonImmunoResearch) for 30 minutes at 4° C. Cells were then filtered andanalyzed on Accuri Flow Cytometer (BD Biosciences, San Jose, Calif.,USA). Unstained and secondary antibody alone controls were also testedfor binding to all cell lines. The results were analyzed using FlowJoversion 9.52 software (Tree Star, Inc., Ashland, Oreg., USA) andgeometric mean of fluorescence for viable cells was determined.Geometric mean (Geom. mean) of fluorescence for each antibody was thennormalized to geometric mean of unstained cells to obtain relativebinding of antibody (binding ratios) per each cell type: HEK293(parental), HEK293/CRE-luc/hAPLNR and HEK293/CRE-luc/MfAPLNR.

Binding ratios for different anti-APLNR monoclonal antibodies are shownin Tables 3 and 4. As shown in Table 3, seven anti-APLNR antibodiesbound to HEK293/CRE-luc/hAPLNR cells with binding ratios ranging from452 to 4098 fold and to HEK293/CRE-luc/MfAPLNR cells with binding ratiosranging from 31 to 1438 fold. The anti-APLNR antibodies tested bound toHEK293 parental cells with binding ratios ranging from 2 to 9 fold. Theanti-mouse IgG secondary antibody alone and mouse IgG (mIgG) controlantibody bound to cells with binding ratios ranging from 1 to 7 fold. Asshown in Table 4, 7 additional anti-APLNR antibodies bound toHEK293/CRE-luc/hAPLNR cells with binding ratios ranging from 2 to 61fold and to HEK293/CRE-luc/MfAPLNR cells with binding ratios rangingfrom 1 to 31 fold. The anti-APLNR antibodies tested bound to HEK293parental cells with binding ratios ranging from 1 to 3 fold. Theanti-human IgG secondary antibody alone and isotype control antibodybound to cells with binding ratios ranging from 1 to 2 fold.

TABLE 3 Binding of anti-APLNR antibodies to HEK293, HEK293/CRE-luc/hAPLNR and HEK293/CRE-luc/MfAPLNR cell lines. Binding Ratio of Geom.Mean to Unstained Cells HEK293 293/Cre-luc/ 293/Cre-luc/ AntibodyParental hAPLNR MfAPLNR H1M9207N 4 2179 1307 H2aM9209N 9 1643 818H2aM9222N 2 452 31 H2aM9227N 4 4098 1438 H2aM9228N 3 1491 108 H2aM9230N3 2938 658 H2aM9232N 6 2857 678 mIgG control 2 7 6 Secondary 2 4 3Antibody alone Unstained 1 1 1

TABLE 4 Binding of anti-APLNR antibodies to HEK293, HEK293/CRE-luc/hAPLNR and HEK293/CRE-luc/MfAPLNR cell lines. Binding Ratio of Geom.Mean to Unstained Cells HEK293 293/Cre- 293/Cre- Antibody Parentalluc/hAPLNR luc/MfAPLNR H4H9092P 3 37 20 H4H9093P 3 61 31 H4H9101P 1 3 2H4H9103P 2 3 2 H4H9104P 1 2 1 H4H9112P 1 2 2 H4H9113P 1 2 1 IsotypeControl 1 2 2 Secondary 1 2 2 Antibody alone Unstained 1 1 1

As shown in Tables 3 and 4, several anti-APLNR antibodies of the presentinvention bind with specificity to the APLNR.

In addition, 13 antibodies with Apelin fused at their N- or C-terminuswere tested for their ability to bind HEK293/CRE-luc/hAPLNR andHEK293/CRE-luc/MfAPLNR cells. As shown in Table 5, H4H9093P with Apelinfused to its N- or C-terminus demonstrated binding toHEK293/CRE-luc/hAPLNR cells with binding ratios ranging from 31 to 151fold and to HEK293/CRE-luc/MfAPLNR cells binding ratios ranging from 16to 54 fold, while the parental antibody, H4H9093P, boundHEK293/CRE-luc/hAPLNR cells with a binding ratio of 61 fold andHEK293/CRE-luc/MfAPLNR cells with a binding ratio of 31 fold. As shownin Table 5, H4H9092P with Apelin fused to its N- or C-terminusdemonstrated binding to HEK293/CRE-luc/hAPLNR cells with binding ratiosranging from 16 to 79 fold and to HEK293/CRE-luc/MfAPLNR cells withbinding ratios ranging from 6 to 31 fold, while the parental antibody,H4H9092P, bound HEK293/CRE-luc/hAPLNR cells with a binding ration of 37fold and HEK293/CRE-luc/MfAPLNR cells with a binding ratio of 20 fold.As shown in Table 5, H4H9209N with Apelin fused to its N-terminusdemonstrated binding to HEK293/CRE-luc/hAPLNR cells with binding ratiosranging from 106 to 121 fold and to HEK293/CRE-luc/MfAPLNR cells withbinding ratios ranging from 43 to 52 fold, while the parental antibody,H4H9209N, bound HEK293/CRE-luc/hAPLNR cells with a binding ratio of 82fold and HEK293/CRE-luc/MfAPLNR cells with a binding ratio of 42 fold.

The antibody-apelin fusions and control antibodies demonstrated bindingto HEK293 parental cells in this assay with binding ratios ranging from2 to 16 fold. Anti-human IgG secondary antibody alone, anti-myc antibodyfused to Apelin at the N-terminus, and the isotype control antibodybound to cells with binding ratio ratios ranging from 1 to 12 fold.

TABLE 5 Binding of antibody-fusion proteins to HEK293, HEK293/CRE-luc/hAPLNR and HEK293/CRE-luc/MfAPLNR cell lines. Binding Ratio of Geom.Mean to Unstained Cells 293/ 293/ Parental Description of HEK293Cre-luc/ Cre-luc/ Antibody Modification Parental hAPLNR MfAPLNR H4H9093PNo modification 3 61 31 H4H9093P- Nter Vk fusion with 2 31 16 1-NVK3(G4S)3 linker H4H9093P- Cter Vk fusion with 3 60 28 2-CVK3 (G4S)3 linkerH4H9093P- Nter VH fusion with 3 130 49 3-NVH3 (G4S)3 linker H4H9093P-Nter VH fusion with 4 140 52 4-NVH0 no linker H4H9093P- Nter VH fusionwith 3 151 54 5-NVH1 G4S linker H4H9093P- Nter VH fusion with 3 139 476-NVH2 (G4S)2 linker H4H9092P No modification 3 37 20 H4H9092P- Nter VHfusion with 2 79 31 1-NVH3 (G4S)3 linker H4H9092P- Nter Vk fusion with 216 6 2-NVK3 (G4S)3 linker H4H9092P- Cter Vk fusion with 2 31 15 3-CVK3(G4S)3 linker H4H9209N No modification 16 82 42 H4H9209N- Nter VH fusionwith 9 106 43 1-NVH0 no linker H4H9209N- Nter VH fusion with 15 107 512-NVH1 G4S linker H4H9209N- Nter VH fusion with 12 121 52 3-NVH2 (G4S)2linker H4H9209N- Nter VH fusion with 14 121 51 4-NVH3 (G4S)3 linkerAnti-myc Nter VH fusion with 1 12 3 9E10 (G4S)3 linker Isotype control 12 2 Secondary Antibody alone 1 2 2 Unstained 1 1 1 As shown in Table 5,several antibody-fusion proteins of the present invention bind withspecificity to the APLNR.

Example 5. Anti-APLNR Antibodies Modulate Cell Signaling Through APLNR

The ability of anti-APLNR antibodies to activate hAPLNR-mediated cellsignaling was measured using a cyclic AMP assay. The hAPLNR is a7-transmembrane G-protein coupled receptor (GPCR). When activated by itsendogenous ligand, Apelin, it inhibits cAMP production suggesting thatit is coupled to inhibitory G-proteins (G_(i)) (Pitkin et al, 2010,Pharmacol. Rev. 62(3):331-342). Apelin is processed into a number ofisoforms from a prepropeptide, and pyroglutamyl Apelin-13,(Pyr¹)Apelin-13 (referred to in this Example as ‘Apelin’) is one of themore potent isoforms known to activate hAPLNR.

A HEK293 cell line was transfected to stably express the full-lengthhuman hAPLNR (amino acids 1-380 of accession number NP—005152.1; SEQ IDNO: 225), along with a luciferase reporter [cAMP response element(CRE,4X)-luciferase-IRES-GFP]. The resulting cell line,HEK293/CRE-luc/hAPLNR, was maintained in DMEM containing 10% FBS, NEAA,pencillin/streptomycin, and 100 μg/mL Hygromycin B.

To test the G_(i)-coupled activation by hAPLNR, HEK293/CRE-luc/hAPLNRcells are seeded onto 96-well assay plates at 20,000 cells/well inOPTIMEM (Invitrogen, # 31985-070) containing 0.1% FBS,pencillin/streptomycin, and L-glutamine and incubated at 37° C. in 5%CO₂ overnight. The next morning, in order to measure hAPLNR activationvia inhibition of Forskolin-induced cAMP, Apelin ((Pyr1)Apelin-13,Bachem, # H-4568) was serially diluted (1:3) from 100 nM to 0.002 nM(including a control sample containing no Apelin), added to cells with 5μM, 7.5 μM, or 10 μM Forskolin (Sigma, # F6886). To measure the abilityof antibodies or Apelin-antibody fusions (see also Example 9) toactivate hAPLNR, antibodies were serially diluted either 1:3 from 500 nMto 0.03 nM, 1:3 from 100 nM to 0.002 nM, 1:4 from 100 nM to 0.0001 nM,or 1:4 from 10 nM to 0.00001 nM, then mixed with 5 μM, 7.5 μM, or 10 μMForskolin, and added to the cells without exogenous Apelin. Testing ofantibodies included a no antibody control. To measure the ability ofantibodies or Apelin-antibody fusions to inhibit hAPLNR, antibodies wereserially diluted at either 1:3 from 500 nM to 0.03 nM or from 100 nM to0.002 nM, 1:4 from 100 nM to 0.0001 nM, or 1:4 from 10 nM to 0.00001 nM,including a no antibody control and incubated with cells for 1 hour atroom temperature. After incubation, a mixture with 5 μM Forskolin and100 μM Apelin was added to cells. Luciferase activity was detected after5.5 hours of incubation at 37° C. and in 5% CO₂ followed by addition ofOneGlo substrate (Promega, # E6051) on a Victor X instrument (PerkinElmer).

The results of all assays were analyzed using nonlinear regression(4-parameter logistics) within Prism 5 software (GraphPad). Activationby the antibodies was calculated as a percentage of the maximumactivation seen in the Apelin dose response. Inhibition by theantibodies was calculated as the difference between the maximum andminimum RLU values for each antibody as a percentage of the RLU range of0-100 μM Apelin.

Unmodified anti-APLNR antibodies were tested for their ability toactivate the hAPLNR by measuring the regulation of Forskolin activationin HEK293/CRE-luc/hAPLNR cells. As shown in Table 6A, 13 out of 14unmodified anti-APLNR antibodies, when tested without Apelin, did notdemonstrate activation of hAPLNR at 100 nM, the highest antibody dosetested, while one antibody, H2aM9227N, demonstrated 12% of maximumApelin activation at 100 nM. As shown in Table 6B, 4 out of 5 unmodifiedanti-APLNR antibodies, when tested without Apelin, demonstratedactivation of hAPLNR with 21 to 52% of maximum Apelin activation at 500nM, the highest antibody dose tested, while one antibody, H2aM9232N, didnot demonstrate any measurable activation of hAPLNR at any concentrationtested. Apelin alone activated hAPLNR with EC₅₀ values ranging from 35μM to 44 μM, as shown in Tables 6A and 6B. None of the isotype controlantibodies demonstrated any activation of hAPLNR.

Unmodified anti-APLNR antibodies were tested for their ability toinhibit hAPLNR by measuring the regulation of Forskolin activation inHEK293/CRE-luc/hAPLNR cells in the presence of 100 μM Apelin. As shownin Tables 6C and 6D, several unmodified anti-APLNR antibodies, whentested in the presence of Apelin, demonstrated inhibition of 26 to 98%of 100 μM Apelin activation (IC₅₀ values ranging from 2.4 nM to >100nM). Three antibodies tested, H2aM9209N, H2aM9222N, and H4H9093Pdemonstrated weak maximum blockade of Apelin ranging from 6 to 11%, butIC₅₀ values could not be determined. Six antibodies tested (H4H9092P,H4H9101P, H4H9103P, H4H9104P, H4H9112P, and H4H9113P) did notdemonstrate any measurable blockade of hAPLNR signaling. Apelin aloneactivated hAPLNR with EC₅₀ values ranging from 41 μM to 44 μM, as shownin Tables 6C and 6D. None of the isotype control antibodies demonstratedany inhibition of hAPLNR.

TABLE 6A Activation of hAPLNR by 100 nM of unmodified anti-APLNRantibodies 3.5E−11 4.4E−11 (at 10 uM Forskolin) (at 5 uM Forskolin) EC₅₀of Apelin with % Activation at % Activation at Forskolin(M) 100 nM mAb100 nM mAb Antibody tested (at 10 uM Forskolin) (at 5 uM Forskolin)H1M9207N No Activation Not tested H2aM9209N No Activation Not testedH2aM9222N No Activation Not tested H2aM9227N 12% Not tested H2aM9228N NoActivation Not tested H2aM9230N No Activation Not tested H2aM9232N NoActivation Not tested H4H9092P Not tested No Activation H4H9093P Nottested No Activation H4H9101P Not tested No Activation H4H9103P Nottested No Activation H4H9104P Not tested No Activation H4H9112P Nottested No Activation H4H9113P Not tested No Activation Isotype control 1No Activation Not tested Isotype control 2 Not tested No Activation

TABLE 6B Activation of hAPLNR by 500 nM of unmodified anti-APLNRantibodies 6.3E−12 EC₅₀ of Apelin with (at 7.5 uM Forskolin) Forskolin(M) % Activation at 500 nM Antibody tested mAb (at 7.5 uM Forskolin)H2aM9222N 21% H2aM9227N 45% H2aM9228N 49% H2aM9230N 52% H2aM9232N NoActivation Isotype control 1 No Activation

TABLE 6C Inhibition of hAPLNR by unmodified anti-APLNR antibodies4.1E−11 (at 5 uM Forskolin) % Inhibition at 100 nM EC₅₀ of Apelin withantibody (IC₅₀ [M]), in the Forskolin (M) presence of 100 ρM Antibodytested Apelin (at 5 μM Forskolin) H1M9207N 83% (3.0E−09) H2aM9209N 7%(IC) H2aM9222N 11% (IC) H2aM9227N 33% (4.3E−09) H2aM9228N 26% (>1.0E−07)H2aM9230N 49% (2.4E−09) H2aM9232N 98% (4.2E−09) Isotype control 1 NoInhibition IC = IC₅₀ value could not be determined

TABLE 6D Inhibition of hAPLNR by unmodified anti-APLNR antibodies4.4E−11 (at 5 uM Forskolin) % Inhibition at 100 nM EC₅₀ of Apelin withantibody, in the presence of Forskolin (M) 100 ρM Apelin (at 5 μMAntibody tested Forskolin) H4H9092P No Inhibition H4H9093P 6% H4H9101PNo Inhibition H4H9103P No Inhibition H4H9104P No Inhibition H4H9112P NoInhibition H4H9113P No Inhibition Isotype control 2 No Inhibition

Example 6. APLNR-Mediated Receptor Signaling by Anti-APLNR Antibodies asMeasured in the pERK Assay

To further measure the effect of anti-APLNR antibodies of the inventionon the APLNR signaling pathway, an assay was used to quantify the amountof phosphorylated ERK1/2 (pERK1/2) and total ERK from an APLNRexpressing cell line (herein referred to as a “pERK assay”). A Chinesehamster ovary (CHO) cell line was transfected to stably express thefull-length human APLNR (hAPLNR; SEQ ID NO: 225). The resulting cellline, CHO/hAPLNR, was maintained in Ham's F12 containing 10% FBS,penicillin/streptomycin, L-glutamine, and 250 μg/mL Hygromycin B.

For the assay, CHO/hAPLNR cells were seeded onto 96 well assay plates at10,000 cells/well in Ham's F12 containing 10% FBS,penicillin/streptomycin, L-glutamine, and 250 μg/mL Hygromycin B. Thenext day, to induce expression of the APLNR and prepare the cells forthe pERK assay, plates were washed and then incubated overnight in Ham'sF12 containing 1% BSA, 0.1% FBS, penicillin/streptomycin, L-glutamineand 0.5 μg/ml doxycycline. After incubation, cells were washed again,and then serial dilutions ranging from 1×10⁻⁶ to 1×10⁻¹³ M of anti-APLNRantibodies, Apelin-13 peptide (Celtek Peptides custom synthesis, Lot#110712), or an isotype control antibody were added to the cells. Cellswere incubated at 37° C. in 5% CO₂ for 15 minutes. Cells were thenwashed and ELISAOne lysis buffer mix (Cat. # EBF001, TGR BioSciences,Adelaide, Australia) was added to the plates and incubated at roomtemperature for 10 minutes while shaking at 300 rpm. Forty μL (40 μL) ofcell lysate was then transferred to each ELISA plate, one to measurepERK1/2 and one to measure total ERK. The ELISAs to detect pERK1/2(ELISAOne # EKT001, TGR BioSciences) and to detect total ERK (ELISAOne #EKT011, TGR BioSciences) were performed as per the manufacturer'sspecifications. The fluorescence signals were then measured using aSpectramax plate reader (Molecular Devices, Sunnyvale, Calif., USA). Theratio of measured pERK1/2 to measured total ERK was calculated and theresults were analyzed using GraphPad Prism software.

As shown in Table 7, two (2) anti-APLNR antibodies tested, H2aM9222N andH2aM9228N, increased the ratio of pERK1/2 to total ERK1/2 with EC₅₀values of 47.61 nM and 64.12 nM, respectively, while Apelin-13 aloneincreased the ratio of pERK1/2 to total ERK1/2 with an EC₅₀ value 38.86μM. The maximum increase in the ratio of pERK1/2 to total ERK1/2 for the2 antibodies was less than that of Apelin-13. One anti-APLNR antibody,H2aM9232N, decreased the ratio of pERK1/2 to total ERK with an IC₅₀value of 208.7 nM.

TABLE 7 Activity of anti-APLNR antibodies and Apelin-13 peptide inpERK/total ERK assays Antibody Activating Inhibiting tested EC₅₀ (M)IC₅₀ (M) H2aM9222N 4.7861E−08  — H2aM9228N 6.412E−08 — H2aM9232N —2.087E−07 Apelin-13 3.886E−11 —

Example 7. Effect of Systemic Administration of an Anti-APLNR AntagonistAntibody (50 mg/kg) in a Blinded Retinal Vascular Development (RVD)Model

To assess the in vivo characteristics of select anti-APLNR antibodies ofthe invention, their ability to block APLNR-mediated angiogenesis in theeye vasculature was measured.

A retinal vascular development (RVD) model was used to evaluate theeffects of an antagonistic anti-APLNR antibody on blood vessel outgrowthin the normal developing retina of mouse pups that were of a mixedbackground strain (75% C57BL6 and 25% Sv129) and homozygous forexpression of human APLNR in place of mouse APLNR (humanized APLNRmice). Pups were subcutaneously injected on postnatal day 2 (P2) witheither 50 mg/kg of an anti-APLNR antagonist antibody, H2aM9232N, or anirrelevant human Fc (hFc) control. Reagents were masked and labeled asSolution A and Solution B to prevent experimenter bias. At postnatal day5, tissue samples were collected and then fixed in PBS containing 4%paraformaldehyde. Fixed tissue samples were washed with PBS three timesfor 15 minutes, and subsequently stained with GS Lectin I (VectorLaboratories, # FL-1101) diluted 1:200 in 1×PBS containing 1% BSA in0.25% Triton-X 100 overnight at 25° C. to visualize retinal vasculature.The following day, stained samples were rinsed with PBS three times for15 minutes each, flat-mounted onto slides, and coverslips weresubsequently mounted using Prolong Gold (Invitrogen, # P36930). Imageswere taken at 20 times magnification using an epi-fluorescent microscope(Nikon Eclipse 80). The vascularized areas in the retina were measuredfrom acquired images from this assay using Adobe Photoshop CS6 extended.Statistical differences between the results obtained from the IgGcontrol antibody and H2aM9232N treated samples were assessed using a twotailed, unpaired Student T-test (**, p<0.001). Only after retinalvasculature area measurements and statistical analysis were completed,the sample identities were unmasked.

TABLE 8 Analysis of the effects of an anti-APLNR antibody in RVD modelRetinal blood vessel outgrowth Antibody Animal # Eye (mm²) IgG controlMouse 1 OD 4.09 antibody Mouse 1 OS 4.44 Mouse 2 OD 3.80 Mouse 2 OS 3.66Mouse 3 OD 3.87 Mouse 3 OS 4.11 Mouse 4 OD 4.95 Mouse 4 OS 3.24 MEAN4.02 SEM 0.18 H2aM9232N Mouse 5 OD 2.49 Mouse 5 OS 3.04 Mouse 6 OD 3.57Mouse 6 OS 3.32 Mouse 7 OD 2.23 Mouse 8 OD 2.36 MEAN 2.84 SEM 0.23

As shown in FIG. 1, a single subcutaneous injection of the antagonisticanti-APLNR antibody, H2aM9232N, produced a statistically significantmean reduction of approximately 30% in retinal blood vessel outgrowth inthe developing mouse retina, indicating that APLNR blockade has asignificant anti-angiogenic effect at postnatal day 5.

As shown in Table 8, eyes harvested from mice injected with theantagonistic anti-APLNR antibody, H2aM9232N, demonstrated retinal bloodvessel growth ranging from approximately 2.23 to 3.57 mm². In contrast,eyes harvested from mice injected with human Fc demonstrated retinalblood vessel growth ranging from approximately 3.24 to 4.95 mm².

Example 8—Potency and Efficacy of Modified Apelin Peptides in a CREAssay

Modified Apelin-13 peptides, such as Apelin-13 peptides having one ormore amino acid(s) deleted from or added to the N-terminus orC-terminus, were tested for their relative potencies with respect toAPLNR activation in a bioassay that was developed to detect theactivation of hAPLNR. (See also PCT International Publication No.WO2014/152955 A1, published on Sep. 25, 2014, which is herebyincorporated by reference.) Various antibody fusion proteins havingApelin-13, or modified Apelin peptides, tethered to the N-terminus orC-terminus of select anti-APLNR antibodies were also made and tested foractivation as shown in Example 9 hereinbelow.

Briefly, an HEK293 cell line was transfected to stably express thefull-length human hAPLNR (amino acids 1-380 of accession numberNP_005152.1), along with a luciferase reporter [cAMP response element(CRE,4X)-luciferase]. The resulting cell line, HEK293/CRE-luc/hAPLNR,was maintained in DMEM containing 10% FBS, NEAA, pencillin/streptomycin,and 100 μg/mL hygromycin B. For the bioassay, HEK293/CRE-luc/hAPLNRcells were seeded onto 96-well assay plates at 20,000 cells/well in 80μL of OPTIMEM supplemented with 0.1% FBS andpenicillin/streptomycin/L-glutamine and incubated for 16 hours at 37° C.in 5% CO2. The next morning, to measure inhibition of forskolin-inducedcAMP production via hAPLNR activation, unmodified apelin peptide andmodified apelin peptides (see Table 9) were serially diluted (1:3) thenmixed with forskolin (Sigma, # F6886) in assay buffer (5 μM finalforskolin concentration), and added to the cells. After 5 hours ofincubation at 37° C. in 5% CO2, luminescence was measured following theaddition of One Glo reagent (Promega, # E6051) using a Victor Xinstrument (Perkin Elmer). The data were fit by nonlinear regression toa 4-parameter logistic equation with Prism 5 software (GraphPad).

As shown in Table 9, apelin-13 can tolerate deletions of amino acidsfrom both the N-terminus and C-terminus while still retaining fullefficacy in the CRE assay, and displaying different degrees of reducedpotency compared to apelin-13. Furthermore, apelin-13 can tolerate theaddition of amino acid residues to its C-terminus, such as five glycineresidues (e.g. apelin-13+5G), and still retain full efficacy but withreduced potency, relative to apelin-13.

TABLE 9 Modified Apelin Peptides Tested in CRE Assay Apelin PeptideAmino Acid Sequence EC₅₀ (M) Apelin-13 QRPRLSHKGPMPF 1.403e−013 (SEQ IDNO: 228) Apelin-F13A QRPRLSHKGPMPA 1.027e−010 (SEQ ID NO: 260)Apelin65-76/Apelin-Cter12 QRPRLSHKGPMP 5.713e−011 (SEQ ID NO: 261)Apelin65-75/Apelin-Cter11 QRPRLSHKGPM 3.604e−012 (SEQ ID NO: 262)Apelin-12 RPRLSHKGPMPF 8.704e−013 (SEQ ID NO: 263) Apelin-11 PRLSHKGPMPF4.379e−010 (SEQ ID NO: 264) Apelin66-76 RPRLSHKGPMP 5.194e−012 (SEQ IDNO: 265) Apelin67-76 PRLSHKGPMP 1.137e−013 (SEQ ID NO: 266) Apelin66-75RPRLSHKGPM 2.174e−012 (SEQ ID NO: 267) Apelin67-75 PRLSHKGPM 3.738e−007(SEQ ID NO: 268) Apelin-13 + 5G QRPRLSHKGPMPFGGGGG 1.469e−010 (SEQ IDNO: 269)

Example 9. Antibody-Fusion Proteins Activate APLNR

The ability of antibody-apelin fusions to activate hAPLNR-mediated cellsignaling was measured using a cyclic AMP assay, similarly to the assaydescribed hereinabove in Example 5. Apelin peptides of various lengthsfused to three different anti-APLNR antibodies (H4H9092P, H4H9093P andH4H9209N) at the N- or C-terminus of the antibodies, and Apelin fused tothe N-terminus of a control antibody (anti-myc), were tested for theirability to activate hAPLNR by measuring the regulation of Forskolinactivation in HEK293/CRE-luc/hAPLNR cells. Several Apelin-antibodyfusions demonstrated activation of hAPLNR with a level of activationsimilar to that of Apelin. The Apelin-antibody fusions had EC₅₀ valuesranging from 27 μM to 29 nM with 10 μM or 7.5 μM Forskolin, as shown inTables 10A and 10B, respectively. Apelin alone activated hAPLNR withEC₅₀ values of 25 μM with 10 μM Forskolin and 39 μM with 7.5 μMForskolin. Two Apelin-antibody fusions, without any linkers, did notdemonstrate any measurable activation of hAPLNR. Furthermore,apelin-cter11, as well as apelin-cter11+serine, fusions induced fullactivation (tested with 7.5 μM Forskolin). However, activation tended todecrease when Apelin peptide was truncated at the C-terminus to 10 aminoacids, and no activation was seen when Apelin length was decreased atits C-terminus to 9 amino acids. Apelin fused to an irrelevant antibody(anti-myc antibody) demonstrated activation of 37% and 53% at 100 nM inseparate experiments, indicating that Apelin fused to an irrelevantantibody activates, but weakly compared with Apelin alone or Apelinfused to anti-APLNR antibodies.

Apelin-antibody fusions were also tested for their ability to inhibithAPLNR by measuring the regulation of Forskolin activation inHEK293/CRE-luc/hAPLNR cells. See Tables 10C and 10D. FiveApelin-antibody fusions demonstrated weak blockade of hAPLNR between 13to 29% at the highest concentration tested. Apelin fused to theN-terminus of an irrelevant antibody (anti-myc antibody) and an isotypecontrol did not demonstrate any measureable inhibition of hAPLNR.

TABLE 10A Activation of hAPLNR by Antibody-Apelin fusions (10 μMForskolin) in the HEK293/CRE-luc/hAPLNR cell line EC₅₀ of Apelin 2.5E−11(10 μM Forskolin) Antibody Apelin % Activation (-fusion) Modification(Fusion) Length at 100 nM mAb tested Description (Sequence) EC₅₀ [M] (10μM Forskolin) H4H9093P No modification No Apelin No No ActivationActivation H4H9093P- Nter Vk fusion with 13 7.7E−09 100% 1-NVK3 (G4S)3linker H4H9093P- Cter Vk fusion with 13 9.2E−11 100% 2-CVK3 (G4S)3linker H4H9093P- Nter VH fusion with 13 5.9E−11 100% 3-NVH3 (G4S)3linker H4H9093P- Nter VH fusion with 13 No No 4-NVH0 no linkerActivation Activation H4H9093P- Nter VH fusion with 13 4.6E−10 100%5-NVH1 G4S linker H4H9093P- Nter VH fusion with 13 8.7E−11 100% 6-NVH2(G4S)2 linker H4H9093P- Nter VH fusion with  9 Not Not APN9- (G4S)3linker (SEQ ID tested tested (G4S)3 NO: 270) H4H9093P- Nter VH fusionwith 10 Not Not APN10- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO:271) H4H9093P- Nter VH fusion with 11 Not Not APN11- (G4S)3 linker (SEQID tested tested (G4S)3 NO: 262) H4H9093P- Nter VH fusion with 11 + SNot Not APN11 + S - (G4S)3 linker (SEQ ID tested tested (G4S)3 NO: 272)H4H9093P- Nter VH fusion with V5-11 Not Not APNV5-11- (G4S)3 linker (SEQID tested tested (G4S)3 NO: 273) H4H9092P No modification No Apelin NoNo Activation Activation H4H9092P- Nter VH fusion with 13 7.3E−11 100%1-NVH3 (G4S)3 linker H4H9092P- Nter Vk fusion with 13 2.9E−08 100%2-NVK3 (G4S)3 linker H4H9092P- Cter Vk fusion with 13 7.5E−10 100%3-CVK3 (G4S)3 linker H4H9209N No modification No Apelin No No ActivationActivation H4H9209N- Nter VH fusion with 13 No No 1-NVH0 no linkerActivation Activation H4H9209N- Nter VH fusion with 13 2.9E−09 100%2-NVH1 G4S linker H4H9209N- Nter VH fusion with 13 6.5E−11 100% 3-NVH2(G4S)2 linker H4H9209N- Nter VH fusion with 13 2.8E−11 100% 4-NVH3(G4S)3 linker H4H9209N- Nter VH fusion with  9 Not Not APN9- (G4S)3linker (SEQ ID tested tested (G4S)3 NO: 270) H4H9209N - Nter VH fusionwith 10 Not Not APN10- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO:271) H4H9209N - Nter VH fusion with 11 Not Not APN11- (G4S)3 linker (SEQID tested tested (G4S)3 NO: 262) H4H9209N - Nter VH fusion with 11 + SNot Not APN11 + S - (G4S)3 linker (SEQ ID tested tested (G4S)3 NO: 272)Anti-myc Nter VH fusion with 13 >1.0E−07   37% 9E10 (G4S)3 linkerIsotype No modification No Apelin No No control 3 Activation Activation

TABLE 10B Activation of hAPLNR by Antibody-Apelin fusions (7.5 μMForskolin) in the HEK293/CRE-luc/hAPLNR cell line EC₅₀ of Apelin 3.9E−11(7.5 μM Forskolin) Antibody Apelin % Activation (-fusion) Modification(Fusion) Length at 100 nM mAb tested Description (Sequence) EC₅₀ [M](7.5 μM Forskolin) H4H9093P No modification No Apelin No No ActivationActivation H4H9093P- Nter Vk fusion with 13 Not Not 1-NVK3 (G4S)3 linkertested tested H4H9093P- Cter Vk fusion with 13 Not Not 2-CVK3 (G4S)3linker tested tested H4H9093P- Nter VH fusion with 13 8.7E−11 100%3-NVH3 (G4S)3 linker H4H9093P- Nter VH fusion with 13 Not Not 4-NVH0 nolinker tested tested H4H9093P- Nter VH fusion with 13 Not Not 5-NVH1 G4Slinker tested tested H4H9093P- Nter VH fusion with 13 Not Not 6-NVH2(G4S)2 linker tested tested H4H9093P- Nter VH fusion with  9 No No APN9-(G4S)3 linker (SEQ ID Activation Activation (G4S)3 NO: 270) H4H9093P-Nter VH fusion with 10 1.4E−09  50% APN10- (G4S)3 linker (SEQ ID (G4S)3NO: 271) H4H9093P- Nter VH fusion with 11 1.6E−10 100% APN11- (G4S)3linker (SEQ ID (G4S)3 NO: 262) H4H9093P- Nter VH fusion with 11 + S9.2E−11 100% APN11 + S- (G4S)3 linker (SEQ ID (G4S)3 NO: 272) H4H9093P-Nter VH fusion with V5-11 5.8E−09 100% APNV5- (G4S)3 linker (SEQ ID11-(G4S)3 NO: 273) H4H9092P No modification No Apelin Not Not testedtested H4H9092P- Nter VH fusion with 13 Not Not 1-NVH3 (G4S)3 linkertested tested H4H9092P- Nter Vk fusion with 13 Not Not 2-NVK3 (G4S)3linker tested tested H4H9092P- Cter Vk fusion with 13 Not Not 3-CVK3(G4S)3 linker tested tested H4H9209N No modification No Apelin No NoActivation Activation H4H9209N- Nter VH fusion with 13 Not Not 1-NVH0 nolinker tested tested H4H9209N- Nter VH fusion with 13 Not Not 2-NVH1 G4Slinker tested tested H4H9209N- Nter VH fusion with 13 Not Not 3-NVH2(G4S)2 linker tested tested H4H9209N- Nter VH fusion with 13 2.4E−11100% 4-NVH3 (G4S)3 linker H4H9209N- Nter VH fusion with  9 No No APN9-(G4S)3 linker (SEQ ID Activation Activation (G4S)3 NO: 270) H4H9209N-Nter VH fusion with 10 1.2E−09  38% APN10- (G4S)3 linker (SEQ ID (G4S)3NO: 271) H4H9209N- Nter VH fusion with 11 2.7E−11 100% APN11- (G4S)3linker (SEQ ID (G4S)3 NO: 262) H4H9209N- Nter VH fusion with 11 + S1.1E−10 100% APN11 + S- (G4S)3 linker (SEQ ID (G4S)3 NO: 272) Anti-mycNter VH fusion with 13 1.1E−08  53% 9E10 (G4S)3 linker Isotype Nomodification No Apelin No No control 3 Activation Activation

TABLE 10C Inhibition of hAPLNR by Antibody-Apelin fusions (10 μMForskolin) in the HEK293/CRE-luc/hAPLNR cell line 2.5E−11 (10 μMForskolin) % Inhibition at EC₅₀ of Apelin 100 nM mAb, in Antibody Apelinthe presence of (-fusion) Modification (Fusion) Length 100 ρM Apelin (attested Description (Sequence) IC₅₀ [M] 10 μM Forskolin) H4H9093P Nomodification No Apelin IC 16% H4H9093P- Nter Vk fusion with 13 No No1-NVK3 (G4S)3 linker Inhibition Inhibition H4H9093P- Cter Vk fusion with13 No No 2-CVK3 (G4S)3 linker Inhibition Inhibition H4H9093P- Nter VHfusion with 13 No No 3-NVH3 (G4S)3 linker Inhibition InhibitionH4H9093P- Nter VH fusion with no 13 IC 29% 4-NVH0 linker H4H9093P- NterVH fusion with 13 No No 5-NVH1 G4S linker Inhibition InhibitionH4H9093P- Nter VH fusion with 13 No No 6-NVH2 (G4S)2 linker InhibitionInhibition H4H9093P- Nter VH fusion with  9 Not Not APN9- (G4S)3 linker(SEQ ID tested tested (G4S)3 NO: 270) H4H9093P- Nter VH fusion with 10Not Not APN10- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO: 271)H4H9093P- Nter VH fusion with 11 Not Not APN11- (G4S)3 linker (SEQ IDtested tested (G4S)3 NO: 262) H4H9093P- Nter VH fusion with 11 + S NotNot APN11 + S- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO: 272)H4H9093P- Nter VH fusion with V5-11 Not Not APNV5-11- (G4S)3 linker (SEQID tested tested (G4S)3 NO: 273) H4H9092P No modification No Apelin IC 5% H4H9092P- Nter VH fusion with 13 No No 1-NVH3 (G4S)3 linkerInhibition Inhibition H4H9092P- Nter Vk fusion with 13 No No 2-NVK3(G4S)3 linker Inhibition Inhibition H4H9092P- Cter Vk fusion with 13 NoNo 3-CVK3 (G4S)3 linker Inhibition Inhibition H4H9209N No modificationNo Apelin 5.9E−09 16% H4H9209N- Nter VH fusion with 13 IC 13% 1-NVH0 nolinker H4H9209N- Nter VH fusion with 13 No No 2-NVH1 G4S linkerInhibition Inhibition H4H9209N- Nter VH fusion with 13 No No 3-NVH2(G4S)2 linker Inhibition Inhibition H4H9209N- Nter VH fusion with 13 NoNo 4-NVH3 (G4S)3 linker Inhibition Inhibition H4H9209N- Nter VH fusionwith  9 Not Not APN9- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO:270) H4H9209N - Nter VH fusion with 10 Not Not APN10- (G4S)3 linker (SEQID tested tested (G4S)3 NO: 271) H4H9209N - Nter VH fusion with 11 NotNot APN11- (G4S)3 linker (SEQ ID tested tested (G4S)3 NO: 262)H4H9209N - Nter VH fusion with 11 + S Not Not APN11 + S - (G4S)3 linker(SEQ ID tested tested (G4S)3 NO: 272) Anti-myc Nter VH fusion with 13 NoNo 9E10 (G4S)3 linker Inhibition Inhibition Isotype No modification NoApelin No No control 3 Inhibition Inhibition IC = IC₅₀ value could notbe determined

TABLE 10D Inhibition of hAPLNR by Antibody-Apelin fusions (7.5 uMForskolin) in the HEK293/CRE-luc/hAPLNR cell line 3.9E-11 (7.5 μMForskolin) % Inhibition at EC₅₀ of Apelin 100 nM mAb, in AntibodyModification Apelin the presence of (-fusion) (Fusion) Length 100 ρMApelin (at tested Description (Sequence) IC₅₀ [M] 7.5 μM Forskolin)H4H9093P No modification No Apelin IC  3% H4H9093P- Nter Vk fusion with13 Not Not 1-NVK3 (G4S)3 linker tested tested H4H9093P- Cter Vk fusionwith 13 Not Not 2-CVK3 (G4S)3 linker tested tested H4H9093P- Nter VHfusion with 13 No No 3-NVH3 (G4S)3 linker inhibition inhibitionH4H9093P- Nter VH fusion with 13 Not Not 4-NVH0 no linker tested testedH4H9093P- Nter VH fusion with 13 Not Not 5-NVH1 G4S linker tested testedH4H9093P- Nter VH fusion with 13 Not Not 6-NVH2 (G4S)2 linker testedtested H4H9093P- Nter VH fusion with  9 1.3E−08 27% APN9- (G4S)3 linker(SEQ ID (G4S)3 NO: 270) H4H9093P- Nter VH fusion with 10 3.2E−08 15%APN10- (G4S)3 linker (SEQ ID (G4S)3 NO: 271) H4H9093P- Nter VH fusionwith 11 No No APN11- (G4S)3 linker (SEQ ID inhibition inhibition (G4S)3NO: 262) H4H9093P- Nter VH fusion with 11 + S No No APN11 + S - (G4S)3linker (SEQ ID inhibition inhibition (G4S)3 NO: 272) H4H9093P- Nter VHfusion with V5-11 No No APNV5-11- (G4S)3 linker (SEQ ID inhibitioninhibition (G4S)3 NO: 273) H4H9092P No modification No Apelin Not Nottested tested H4H9092P- Nter VH fusion with 13 Not Not 1-NVH3 (G4S)3linker tested tested H4H9092P- Nter Vk fusion with 13 Not Not 2-NVK3(G4S)3 linker tested tested H4H9092P- Cter Vk fusion with 13 Not Not3-CVK3 (G4S)3 linker tested tested H4H9209N No modification No Apelin NoNo inhibition inhibition H4H9209N- Nter VH fusion with 13 Not Not 1-NVH0no linker tested tested H4H9209N- Nter VH fusion with 13 Not Not 2-NVH1G4S linker tested tested H4H9209N- Nter VH fusion with 13 Not Not 3-NVH2(G4S)2 linker tested tested H4H9209N- Nter VH fusion with 13 No No4-NVH3 (G4S)3 linker Inhibition Inhibition H4H9209N- Nter VH fusion with 9 9.3E−09 14% APN9- (G4S)3 linker (SEQ ID (G4S)3 NO: 270) H4H9209N -Nter VH fusion with 10 No No APN10- (G4S)3 linker (SEQ ID InhibitionInhibition (G4S)3 NO: 271) H4H9209N - Nter VH fusion with 11 No NoAPN11- (G4S)3 linker (SEQ ID inhibition inhibition (G4S)3 NO: 262)H4H9209N - Nter VH fusion with 11 + S No No APN11 + S - (G4S)3 linker(SEQ ID inhibition inhibition (G4S)3 NO: 272) Anti-myc Nter VH fusionwith 13 No No 9E10 (G4S)3 linker inhibition inhibition Isotype Nomodification No Apelin No No control 3 Inhibition Inhibition

Example 10: Activation of APLNR-Mediated Receptor Signaling byAntibody-Fusion Proteins in the pERK Assay

Experiments were done as essentially shown in Example 6, describedhereinabove. As shown in Table 11, two (2) antibody-Apelin fusions ofthe invention increased the ratio of pERK1/2 to total ERK1/2 with EC50values of 542.2 μM and 271.4 μM, while Apelin-13 increased the ratio ofpERK1/2 to total ERK1/2 with an EC50 value of 32.48 μM.

TABLE 11 Activity of antibody-Apelin fusions and Apelin-13 peptide inpERK/total ERK assays Sample tested Activating EC₅₀ (M) H4H9093P Nter VHfusion with (G4S)3 linker 5.422E−10 (H4H9093P-3-NVH3) H4H9209N Nter VHfusion with (G4S)3 linker 2.714E−10 (H4H9209N-4-NVH3) Apelin-13 (nofusion) 3.248E−11

11: Activation of APLNR-Mediated Receptor Signaling by Antibody-FusionProteins in a β-Arrestin Assay

A PathHunter® eXpress AGTRL1 CHO-K1 β-Arrestin GPCR cell based assay(DiscoverX, # 93-0250E2) was used to assess signaling throughrecruitment of β-Arrestin by the activated human Apelin receptor(hAPLNR). To test the β-arrestin recruitment upon hAPLNR activation,PathHunter® eXpress AGTRL1 CHO-K1 β-Arrestin cells were seeded onto96-well assay plates at 8500 cells/well according to the manufacturer'sprotocol and incubated at 37° C. in 5% CO₂ for two nights. On the day ofthe assay, Apelin, pyroglutamyl Apelin-13, (Bachem, # H-4568) wasserially diluted (1:3) from 500 nM to 0.08 nM (including a controlsample containing no Apelin) and added to the cells.

To measure the ability of Apelin-antibody fusions and antibodies toactivate hAPLNR, Apelin-antibody fusions and antibodies were seriallydiluted (1:3) from 500 nM to 0.08 nM and added to the cells withoutexogenous Apelin. Testing of Apelin-antibody fusions and antibodiesincluded a no antibody control. After 1.5 hours of incubation at 37° C.in 5% CO₂, chemiluminescent activity was detected on a Victor Xinstrument (Perkin Elmer) after an addition of PathHunter® DetectionReagents.

The results of all assays were analyzed using nonlinear regression(4-parameter logistics) within Prism 5 software (GraphPad). Activationby the antibodies and Apelin-antibody fusions was calculated as apercentage of the maximum activation seen in the Apelin dose response.In the PathHunter® eXpress AGTRL1 CHO-K1 β-Arrestin cell based assay,all 11 of the anti-APLNR antibodies fused to Apelin peptides testeddemonstrated partial activation of hAPLNR with activation ranging from2-64% of maximum Apelin activation, and corresponding EC₅₀ valuesranging from 970 μM to >100 nM. Anti-APLNR antibodies without Apelinfusion showed little to no activation. Apelin activated hAPLNR with anEC₅₀ value of 1.5 nM. Apelin fused to an irrelevant anti-myc antibodydemonstrated weak activation of hAPLNR at 6% at the highestconcentration tested 500 nM, without a measurable EC₅₀, while an isotypecontrol antibody did not demonstrate any measurable activation in thisassay.

TABLE 12 Activation of hAPLNR by Antibody-Apelin fusions and antibodiesin PathHunter ® eXpress AGTRL1 CHO-K1 β-Arrestin cell based assayPathHunter ® eXpress AGTRL1 CHO-K1 Cell Line Tested: β-Arrestin CellsEC₅₀ of Apelin (M): 1.5E−09 Antibody-Apelin fusion description AntibodyModification Apelin % Activation at 500 nM (-fusion) (Fusion) Lengthantibody or Apelin- tested Description (Sequence) EC₅₀ [M] antibodyfusion H4H9093P No modification No Apelin IC  2% H4H9093P- Nter VHfusion with 13 2.1E−09 54% 3-NVH3 (G4S)3 linker H4H9093P- Nter VH fusionwith  9 8.3E−09  4% APN9- (G4S)3 linker (SEQ ID (G4S)3 NO: 270)H4H9093P- Nter VH fusion with 10 6.5E−09  5% APN10- (G4S)3 linker (SEQID (G4S)3 NO: 271) H4H9093P- Nter VH fusion with 11 5.8E−09 64% APN11-(G4S)3 linker (SEQ ID (G4S)3 NO: 262) H4H9093P- Nter VH fusion with 11 +S 2.7E−09 37% APN11 + S- (G4S)3 linker (SEQ ID (G4S)3 NO: 272) H4H9093P-Nter VH fusion with V5-11  >1E−07 47% APNV5-11- (G4S)3 linker (SEQ ID(G4S)3 NO: 273) H4H9209N No modification No Apelin No No ActivationActivation H4H9209N- Nter VH fusion with 13 1.5E−09 30% 4-NVH3 (G4S)3linker H4H9209N- Nter VH fusion with  9 3.5E−09  3% APN9- (G4S)3 linker(SEQ ID (G4S)3 NO: 270) H4H9209N- Nter VH fusion with 10 4.0E−09  2%APN10- (G4S)3 linker (SEQ ID (G4S)3 NO: 271) H4H9209N- Nter VH fusionwith 11 9.7E−10 44% APN11- (G4S)3 linker (SEQ ID (G4S)3 NO: 262)H4H9209N- Nter VH fusion with 11 + S 3.1E−09 25% APN11 + S- (G4S)3linker (SEQ ID (G4S)3 NO: 272) Anti-myc Nter VH fusion with 13 IC  6%9E10 (G4S)3 linker Isotype No No control Activation Activation antibodyIC = EC₅₀ value could not be determined

Example 12—Antibody-Apelin-11 Fusions Show Increased Stability in Serum

To measure the stability and activity of the apelin peptide fusionantibody in serum, fusion antibodies were exposed to mouse serum fordifferent times (0, 6, 24 hours). After antibody purification fromserum, samples were analyzed by mass spectrometry, to evaluate thepresence of apelin fragments. To test activity of the exposedApelin-antibody fusions, the diluted serum with unpurified antibodyfusion was also tested in a beta-arrestin activity assay. Serum obtainedfrom a male C57bl/6 mouse was diluted with PBS at a 1 to 1 ratio. Atotal of 100 μg apelin-antibody fusion was added to serum. 250 ul or 25%of this mixture was removed immediately and placed at −20° C. (t=0timepoint). The remaining mixture was placed in an incubator at 37° C.and 250 μl of the mixture was removed after 6 and 24 hours.

Sample purification via protein A beads: Dynabead™ Protein A beads(Invitrogen Cat # 10001D) were washed 3 times with PBS. 25 μl Dynabead™slurry was added to 225 μl of serum and apelin-antibody fusion mixture.The new mixture was incubated at 4° C. with rotation for 3 hours toallow the antibody fusion binding to the protein A beads. Afterincubation, beads were washed 3 times with PBS. Sixty μl of Laemillidye/buffer was added to the washed and pelleted protein A beads. Thismixture was incubated at 90° C. for 5 minutes, to dissociate thepurified antibody from the protein A beads.

Mass Spectrometry Preparation: Five (5) μl of beta-mercaptoethanol wasadded to the purified antibody and denatured at 95° C. for 10 min. Theentire volume of the purified antibody was loaded onto a Tris-glycinegel and ran at 150V for 1 hr. Coomaise blue was used to stain the gel.The 50 kDa band, corresponding to the heavy chain fragment, was cut outfrom the gel and chopped finely. The excised bands were split into two500 μl tubes. 100 μl of 100 mM ammonium bicarbonate/50% acetonitrile wasadded and incubated at 37° C. for 1 hr to destain the gels. Destainingsolution is removed and 100 μl of 100% acetonitrile is added todehydrate the gel for 5 minutes at ambient temperature. Dehydrationsolution is removed from the gel and 75 μl of 10 mM DTT in 50 mMammonium bicarbonate is added and incubated at 37° C. for 30 minutes toreduce the protein. Reducing solution is removed and 75 μl of 55 mMIodoacetamide in 50 mM ammonium bicarbonate is added and incubated atambient temperature in the dark to alkylate the protein. Alkylationsolution is removed and 100 μl of 50 mM ammonium bicarbonate is added towash the gel. Wash solution is removed and 100 μl of 100% acetonitrileis added to dehydrate the gel for 5 minutes at ambient temperature.Dehydration solution is removed and 30 μl of LYS-C enzyme mixture isadded and digested overnight at 37° C. After overnight digestion samplesare purified with a ZipTip filter in a 10 mg/mlalpha-cyano-4-hyrdoxycinnamic acid/70% acetonitrile/0.1% TFA solutionand spot eluted onto a MALDI target and read on mass spectrometer.

Mass Spectrometry Analysis: The apelin peptide is fused to theN-terminal portion of the APLN-R antibody. The Lys-C recognizes anddigests proteins at the C-terminal side of the amino acid lysine. Thepeptide of interest, after Lys-C digestion of the fusion antibody, hasthe sequence of QRPRLSHK (amino acid residue numbers 1 to 8 of SEQ IDNO: 228), reporting a mass charge ratio peak at 1004.

TABLE 13 Serum stability test at 0, 6, and 24 hours to identify intactfusion by mass spectrometry measurement (peptide fragment peak at 1004)Modification Fusion (Fusion) 0 Hour 6 Hour 24 Hour Tested DescriptionStability Stability Stability H4H9093P-3- Nter APN13 YES NO NO NVH3 with(G4S)3 linker H4H9209N - Nter APN- YES YES NO APN11 + S-(G4S)3 Cter11 +S with (Weak) (G4S)3 linker H4H9209N - Nter APN- YES YES YESAPN11-(G4S)3 Cter11 with (G4S)3 linker H4H9209N - Nter APN- YES YES NOAPN10-(G4S)3 Cter10 with (Weak) (G4S)3 linker H4H9209N- Nter APN-Cter9YES YES YES APN9-(G4S)3 with (G4S)3 (weak) linker

As shown in Table 13, the truncated apelin fusion antibodies reportintact apelin peptide peaks on mass spectrometry after 6 hours of serumexposure. The apelin-cter11 fusion antibody has residual apelin peakafter 24 hours of serum exposure. See also FIG. 2.

To test activity of the exposed Apelin-antibody fusions, the dilutedserum with unpurified antibody fusion (H4H9093P-3-NVH3,H4H9209N-APN11-(G4S)3, or H4H9209N-APN11+S-(G4S)3) was tested in abeta-arrestin activity assay, as described above in Example 11 (protocolbased on the DiscoverX B-Arrestin activity assay kit). The treatmentconcentration of each unpurified Apelin-antibody fusion was 1 μg/mL.

Antibody fusions having Apelin-Cter11 and Apelin-Cter11+S at theirC-termini retain β-Arrestin activity even after 6 h of serum exposure.The results of β-Arrestin activity at timepoints 0, 6 and 24 hours aredepicted in FIG. 3. The 6 h timepoint value represents percentactivation relative to the 0 h timepoint, or 2.4%, 70.4% and 33.6% forH4H9093P-3-NVH3, H4H9209N-APN11-(G4S)3, or H4H9209N-APN11+S-(G4S)3,respectively.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method of treating cancer or metastaticdisease, comprising administering, to a patient in need thereof, atherapeutically effective amount of an isolated antibody orantigen-binding fragment thereof that binds to apelin receptor (APLNR)and blocks the interaction of APLNR and apelin, wherein the antibody orantigen-binding fragment comprises: (a) the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,34, 50, 66, 82, 98, and 130; and (b) the CDRs of a light chain variableregion (LCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, and
 138. 2. Themethod of claim 1, wherein the antibody or antigen-binding fragmentcomprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively,selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16;20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64;68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112; and132-134-136-140-142-144.
 3. The method of claim 1, wherein the antibodyor antigen-binding fragment comprises: (a) a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, and 130; and (b) a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, and
 138. 4.The method of claim 3, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, and 130/138.
 5. The method of claim 4, wherein the antibody orantigen-binding fragment comprises the HCVR/LCVR amino acid sequencepair of SEQ ID NOs: 98/106.
 6. The method of claim 4, wherein theantibody or antigen-binding fragment comprises the HCVR/LCVR amino acidsequence pair of SEQ ID NOs: 2/10.
 7. The method of claim 4, wherein theantibody or antigen-binding fragment comprises the HCVR/LCVR amino acidsequence pair of SEQ ID NOs: 18/26.
 8. The method of claim 4, whereinthe antibody or antigen-binding fragment comprises the HCVR/LCVR aminoacid sequence pair of SEQ ID NOs: 130/138.
 9. The method of claim 1,wherein the antibody or antigen-binding fragment comprises the CDRs of aHCVR/LCVR amino acid sequence pair of SEQ ID NOs: 98/106.
 10. The methodof claim 1, wherein the antibody or antigen-binding fragment comprisesthe CDRs of a HCVR/LCVR amino acid sequence pair of SEQ ID NOs: 2/10.11. The method of claim 1, wherein the antibody or antigen-bindingfragment comprises the CDRs of a HCVR/LCVR amino acid sequence pair ofSEQ ID NOs: 18/26.
 12. The method of claim 1, wherein the antibody orantigen-binding fragment comprises the CDRs of a HCVR/LCVR amino acidsequence pair of SEQ ID NOs: 130/138.
 13. The method of claim 1, whereinthe antibody or antigen-binding fragment comprises the CDRs of aHCVR/LCVR amino acid sequence pair selected from the group consistingof: SEQ ID NOs: 34/42, 50/58, 66/74, and 82/90.
 14. A method of treatingretinopathy, comprising administering, to a patient in need thereof, atherapeutically effective amount of an isolated antibody orantigen-binding fragment thereof that binds to apelin receptor (APLNR)and blocks the interaction of APLNR and apelin, wherein the antibody orantigen-binding fragment comprises: (a) the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,34, 50, 66, 82, 98, and 130; and (b) the CDRs of a light chain variableregion (LCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, and
 138. 15. Themethod of claim 14, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, and 130/138.
 16. The method of claim 14, wherein the antibody orantigen-binding fragment comprises the CDRs of a HCVR/LCVR amino acidsequence pair of SEQ ID NOs: 98/106.
 17. The method of claim 14, whereinthe antibody or antigen-binding fragment comprises the HCVR/LCVR aminoacid sequence pair of SEQ ID NOs: 98/106.
 18. A method of treatingpathological angiogenesis, comprising administering, to a patient inneed thereof, a therapeutically effective amount of an isolated antibodyor antigen-binding fragment thereof that binds to apelin receptor(APLNR) and blocks the interaction of APLNR and apelin, wherein theantibody or antigen-binding fragment comprises: (a) the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR)having an amino acid sequence selected from the group consisting of SEQID NOs: 2, 18, 34, 50, 66, 82, 98, and 130; and (b) the CDRs of a lightchain variable region (LCVR) having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, and138.
 19. The method of claim 18, wherein the antibody or antigen-bindingfragment comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, selected from the group consisting of: SEQ ID NOs:4-6-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64;68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112; and132-134-136-140-142-144.
 20. The method of claim 18, wherein theantibody or antigen-binding fragment comprises a HCVR/LCVR amino acidsequence pair selected from the group consisting of: SEQ ID NOs: 2/10,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, and 130/138.